CRISPR Cas Bacterial Adaptive Immunity

CRISPR Cas:
From Bacterial Adaptive Immunity to a

Genome Editing Revolution
By Rodolphe Barrangou
A Narrative produced by The Explorer’s Guide to Biology

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The Explorer’s Guide to Biology
https://explorebiology.org/
CRISPR-Cas
From Bacterial Adaptive Immunity to a Genome Editing Revolution
Rodolphe Barrangou, North Carolina State University
Dr. Barrangou
Dr. Barrangou spent 9 years in R&D at Danisco and DuPont in the
Food Industry, and is the T. R. Klaenhammer Distinguished Professor
in Probiotics Research in the Department of Food, Bioprocessing and
Nutrition Sciences at North Carolina State University. He teaches an
introductory undergraduate course on bioprocessing (the use of biolog-
ical processes and materials to manufacture products) and biopharma-
ceutical sciences. His lab focuses on the functions of
CRISPR-Cas systems, and their applications in bacteria
used in food manufacturing. Rodolphe has received
several awards for this work on CRISPR, including the
Canada Gairdner Award, and has been involved in several start-up companies
using CRISPR-based technologies.
Version Date: September, 2019
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CRISPR-Cas: From Bacterial Adaptive Immunity to a Genome Editing Revolution
Summary
Humans are plagued by viruses, which cause illnesses from the common cold to AIDS. However,
humans are not unique in their need to fight viruses. Even bacteria are preyed upon by viruses
called bacteriophages, which are the most ubiquitous and abundant biological entity on our
planet. Viruses and bacteria are engaged in an evolutionary arms-race for survival, which has
given rise to a diverse set of defense systems that enable bacteria to evade phage predation.
Similar to adaptive immunity in humans, bacteria can capture and store a molecular memory of a
prior encounter with a virus in their genomes, and this “vaccination” allows bacteria to seek and
destroy the nucleic acids of subsequent related invaders. The discovery of this bacterial immune
system, called CRISPR-Cas, emerged unexpectedly from putting together clues about mysterious
DNA sequences in bacterial genomes and from the need of the food industry to keep bacterial
yoghurt and cheese cultures free of viral infection. After this discovery, scientists learned how to
repurpose the molecular machinery from these immune systems to edit the sequence of genomes,
including those of humans. In addition to creating a new research technology that is sweeping the globe,
the ability to rewrite genomes has monumental implications for medicine (human gene therapy),
agriculture (next-generation plants and livestock), biotechnology (engineering of microbes for the
synthesis of bioproducts), and beyond.
Learning Overview
Big concepts
The CRISPR-Cas bacterial immune system allows bacteria to selectively remember the nucleic acid
sequences of prior viral invaders and defend themselves against future viral attacks. Scientists
have exploited knowledge of the molecular machines involved in the bacterial immune system
to develop one of the most powerful tools ever created for biotechnology—the ability to edit the
genome of any organism.
Terms and Concepts Used

B Cell, Bacteria, Bacterial Colonies, Bacterial Strain, Bacteriophage, Coding and Non-Coding Regions,
DNA, Enzyme, Fermentation, Gene, Genome, Haploid/Diploid, Homologous Sequence, HIV, Immunity,
Infection, Locus, Microbiome, Pathogen, Phage, Plasmid, Prokaryotes, Proteins, Reading Frame,
Replication, RNA, RNA interference, T Cell, Transcription, Virus, Vaccine
Terms and Concepts Explained

Bioinformatics, CRISPR, Cas, Duchenne Muscular Dystrophy, Genome Editing, Gene Knockout,
Single-Guide RNA, Nuclease
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Introduction
Bacteria are invaded and destroyed by bacteriophage (phage) and have evolved mechanisms
to protect themselves.
This Narrative describes the discovery, mechanism, and applications of one such bacterial
defense system, CRISPR-Cas.
Part I: Journey to Discovery—CRISPR-Cas systems provide adaptive immunity

against phages in bacteria.
Microbes have been used to produce food products through fermentation for millennia.
This Journey to Discovery was stimulated by a very pragmatic problem faced by a food company
called Danisco, Inc. How can bacterial starter cultures for yoghurt be protected against viruses
(bacteriophage)? And why did some bacteria appear to be resistant to viral attack?
Scientists discovered a peculiar region in the bacterial genome with a nucleotide sequence that
was repeated over and over again; they later called it the Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR), but its function remained mysterious for decades.
An intriguing gene called Cas9 was found adjacent to CRISPR. Bioinformatics suggested a
possible function of the gene in the interference of nucleic acid function.
In between the CRISPR repeats were variable nucleotide sequences called “spacers.” Originally
thought to be random and uninteresting, scientists discovered that the spacer sequences were
similar or identical to sequences found in bacteriophages.
Scientists at Danisco, Inc. had access to a large amount of sequence data of different yoghurt
culture strains of bacteria and the phage to which they were exposed. They found that the
CRISPR spacer sequences in the bacteria were similar or identical to the sequences of phage
that attacked them. This finding suggested that perhaps CRISPR plays a role in acquiring
immunity to attack by a particular phage.
Scientists now performed experiments to test the hypothesis that CRISPR plays a role in phage immu-
nity. They challenged bacteria with a particular phage strain. Survivors of the attack had acquired new
CRISPR spacer and those spacers matched the sequence of the phage that attacked them.
They then intentionally manipulated the spacer region by genetic engineering with the follow-
ing outcomes: 1) Adding a spacer, phage resistance was acquired; 2) removing a spacer, resis-
tance was lost; 3) swapping spacers between two strains, their respective phage resistance
was switched too.
The scientists also tested the function of the Cas9 gene. If the gene was “knocked out,” phage
resistance was lost.
These experiments established the biological function of CRISPR-Cas systems as providing
sequence-specific adaptive immunity against viruses in bacteria.
Shortly thereafter, several groups showed that the CRISPR locus produces RNAs and that Cas9
is a nuclease that, under the instructions of the CRISPR RNAs, targets and cleaves the bacte-
riophage DNA, destroying its replication potential.
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Part II: Knowledge overview—How widespread adaptive immunity in bacteria and

archaea enables flexible targeting of nucleic acids.
CRISPR-Cas9 provides an immune system for bacteria. The memory of a prior phage attack is
maintained by storing a small segment of the phage DNA sequence in the bacterial genome
(at the CRISPR locus). The Cas9 enzyme uses this information to cleave the DNA of the same
phage if it tries to invade again.
There are three main steps in the bacterial immune response. In the first step (acquisition), a
segment of the invasive DNA is copied (creating a “spacer”) and then inserted along with a new
CRISPR repeat at the end of the CRISPR array.
In the second step (expression), the CRISPR array is transcribed into a long precursor CRISPR
RNA molecule, which is then cleaved into small CRISPR RNAs (crRNAs).
In the third step (interference), a crRNA and a second RNA (tracrRNA) form a complex with the
protein Cas9. The Cas9-RNA complex scans along DNA randomly; if it finds a sequence that
matches the crRNA, it stops and then cleaves the target DNA.
The Cas9 enzyme has been turned into a programmable genome editing tool. Also see the Key
Experiment on CRISPR-Cas9 by Doudna.
Cells will repair a double-strand break in DNA using two types of DNA repair: non-homologous
end joining (NHEJ) and homology-directed repair (HDR).
CRISPR-Cas9 can be programmed to create a double-strand break at a specific sequence in the
genome. Scientists can also introduce a “repair template” that the repair machinery can use to
repair the break. The repair template can be designed in such a way that a new sequence (an
“edit”) is introduced into the genome during repair.
CRISPR-Cas9 editing has many applications. It can be used to change one (or a few) nucleotide(s)
to another (e.g., to correct a disease mutation), insert a new piece of DNA, or delete a piece of DNA.
The steps in the genome-editing experiment are described.
Part III: Frontier – CRISPR-based molecular machines are powerful genome editing
technologies that enable flexible alteration of genomes and transcriptomes.
CRISPR-based technologies have democratized genome editing and can be easily implemented
in animal, plant, and microbial cells across the tree of life with implications in medicine, agricul-
ture, and biotechnology.
Duchenne Muscular Dystrophy (DMD), a severe disease of muscle degeneration, is a recessive
genetic disorder arising from a mutation in a gene on the X chromosome. The disease occurs
primarily in boys due to their single copy of the X chromosome.
Dogs with a similar DMD mutation are being used as a model to study and treat the disease.
A study is presented in which a virus is used to deliver Cas9, a sgRNA, and a DNA repair tem-
plate to the muscle of dogs with this disease.
Results show that the defective DMD gene in many, but not all, of the muscle cells is corrected
by CRISPR-Cas9 and a healthy appearance of the muscle is restored.
Examples of how CRISPR-Cas9 genome editing is being used in agriculture and livestock are
discussed.
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Closing Thoughts
CRISPR is a great example of how powerful new technologies emerge from asking basic questions
about how life works, in this case, starting with observations of mysterious sequences in the genome
of bacteria.
The ability to edit genomes with ease through CRISPR-Cas technologies raises many important
ethical questions on how this technology should be used in humans and other species.
Guided Papers:
Barrangou, R., et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science
2007;315:1709–1712.
Amoasli, L. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.
Science 2018;362:86–91.
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Introduction
As humans, we are mindful of the many viruses that surround us. Our history is plagued with
catastrophic viruses, and recent viral outbreaks are a reminder of their impact on human health,
including disabling or life-threatening diseases (e.g., HIV and flu), persistent infection (e.g., herpes
and papillomavirus), food poisoning (e.g., norovirus), and animal diseases (e.g., H1N1). All living
organisms are subject to predation by viruses, which are the most numerous biological entities on
the planet. Indeed, viruses can be detected in all environmental conditions and niches, sometimes
in astonishingly high numbers: a sip of ocean water contains more viruses than the number of humans
on Earth. Bacteria suffer the greatest sheer number of viral attacks due to their large numbers.
The viruses that attack bacteria are called bacteriophage (or phage).
Like other organisms, viruses have a genome consisting of nucleic acid (DNA or RNA), which is
surrounded by a protein shell (the capsid) and sometimes by a membrane. However, the differ-
ence between viruses and other living organisms is that viruses neither can replicate on their own
nor do they carry out metabolic activities. Rather, they must invade a host cell and subvert some
of the host machinery for the replication and manufacturing of more viruses (Figure 1). A single
infection typically yields dozens of new viruses from a single host. The compact machinery and
rapid replication of viruses makes them robust, efficient, and fast-evolving molecular machines.
Bacteriophages are also deadly to their hosts: to release their new copies, phage usually lyse and
ultimately kill the bacterium.
As you might imagine from their predator–prey relationship, viruses and their hosts are engaged
in an evolutionary arms race for survival. While viruses try to gain command of the host machin-
ery, hosts develop an arsenal of defense and immune systems that enable them to overcome viral
predation. Indeed, viruses need to evolve quickly to adapt to their host, while the host needs to
mount an adaptive response to prevent viral predation or escape viral infection. Given the pace
at which bacteria grow and replicate (a single bacterium can become a billion overnight ), the
bacteria-phage arms race is unraveling at the most frenetic pace and scale of all predator–prey
relationships on earth.
viruses and their hosts are engaged in an evolutionary arms race for survival
In humans, the adaptive immune response is a powerful way of fighting viral (or bacterial) infec-
tion. For example, a flu vaccine delivers an inactive virus and some of your white blood cells retain
a memory of this viral signature. Consequently, these cells are ready to be quickly mobilized in
case the real (or a closely related) virus shows up later. (It is worth noting that mutations rapidly
change the DNA sequence of the flu virus, which is why flu vaccines are only partially effective.)
For many years, scientists considered bacteria to be simple and incapable of mounting a specific
immune response to viruses. However, we have had a long track record of underestimating and
under-appreciating the sophistication of bacteria (see also the Narrative on Quorum Sensing by
Bassler). This Narrative is about a bacterial adaptive immune system called CRISPR-Cas.
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Figure 1. Phage attacking and replicating inside of a bacterium. In this case, the phage T4 is shown
invading and replicating inside of the E. coli.
In the Journey to Discovery, I will trace the key discoveries that led to our understanding of CRISPR-
Cas as a bacterial immune system. The journey, like so many in science, involved several investi-
gators contributing various clues and pieces of the puzzle. CRISPR stands for Clustered Regularly
Interspaced Short Palindromic Repeats, which is a mouthful for describing a mysterious set of
repeated nucleotide sequences. Although these repeating sequences showed up in the genomes
of many prokaryotes, their function remained elusive for nearly two decades after they were ini-
tially observed. Eventually, a series of clues led several inquisitive groups to posit that they may
constitute a part of an immune system against viruses. This hypothesis naturally led to experi-
ments that my group conducted, demonstrating that CRISPR-Cas provides adaptive immunity in
bacteria.
In the Knowledge Overview, I will explain how several types of CRISPR-based immune systems in
bacteria work. When bacteria encounter a viral attack, they acquire a piece of viral DNA to vac-
cinate them against future attacks by similar viruses. These systems can then thwart subsequent
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attacks by selectively cleaving invasive nucleic acid sequences that match the piece of DNA
acquired during the vaccination process. I will discuss how various CRISPR-Cas systems gen-
erate different types of damage in DNA and RNA. I will also explain how these machines can
be reprogrammed and transplanted into other organisms for flexible targeting and cleavage of
DNA, and how the resulting DNA breaks can be repaired in ways that enable the rewriting of the
DNA sequence in a genome (known as genome editing). I also encourage you to read the Key
Experiment by Doudna, which describes a critical result that opened up the possibility of creating
a programmable CRISPR-Cas system for editing any sequence in the genome. In the Frontiers
section, I will discuss how CRISPR technologies are being exploited by thousands of scientists to
alter the genomes of animals, plants, and microbes and will pick a couple of examples to highlight
this exciting new technology and its impact on medicine, agriculture, and biotechnology.
From what started as curiosity about strange sequences in bacteria, CRISPR has revolutionized
biology in the past decade and has now become a household word. The implications of CRISPR-
fueled genome editing on medicine, agriculture, and biotechnology are staggering. With this pow-
erful new technology comes profound implications for society, encompassing ethical concerns,
regulatory processes, and industrial challenges, which I will return to in my Closing Thoughts. In
addition to shedding light on how we came upon this disruptive discovery and how it is impacting
many fields of science, this Narrative also reveals how science relies on the contributions of many
individuals and how a scientific journey proceeds with a blend of eureka moments and mistakes,
all critical to the scientific process itself.
CRISPR has revolutionized biology in the past decade and has now become a
household word
Part I: The Journey to Discovery

CRISPR-Cas Systems Provide Adaptive Immunity in Bacteria
The Problem
Eating is perhaps the most basic human need—we have domesticated the land and oceans, culti-
vating many plant and animal species throughout history to feed ourselves (also see Narrative on
Plant Genetics by Ronald). Despite all of our scientific knowledge and technological advances, we
still struggle to feed humanity a plentiful, safe, tasty, and health-promoting diet. Although most
people are very familiar with food as a product and a commodity that we consume several times a
day (hopefully), consumers typically have a very limited understanding of where their food comes
from, let alone how it is made or scientifically formulated. Indeed, humankind used fermentation
for millennia to preserve and process foods without any understanding of the microbiological
organisms and processes that drive it. Microbes actively assist in the preservation and genesis of
enjoyable products, including converting grapes into wine, apples into cider, barley into beer, milk
into cheese, cabbage into sauerkraut, or cucumbers into pickles. For more than a century, Louis
Pasteur and subsequent food microbiologists have been working on harnessing microbes for the
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genesis of tasty foods. However, occasionally, fermenting organisms also get infected by viruses
with sometimes dire consequences.
In 2005, I was an industrial food scientist at Danisco, Inc., and we had a problem that was costing
our company time and money. Yoghurt and cheese are made through fermentation of milk by starter
cultures of bacteria and maintaining healthy cultures is the heart of a successful business (Figure 2).
at Danisco, Inc., and we had a problem that was costing our company time and
money
However, many of our yoghurt cultures were occasionally dying, victims of bacteriophage that
can infect and kill the bacteria. However, some of the bacteria cultures survived. Why was it
that some bacteria survived while others succumbed to the virus? The answer to this question
could have very practical consequences for our company, which provided starter cultures to the
dairy industry, and our customers, large yoghurt and cheese industrial manufacturers. Rather
than addressing a specific fundamental question or hypothesis, the practicality of the issue at
hand provided a sense of urgency and a real industrial problem to solve—how do some bacteria
survive phage attack?
Figure 2. The role of bacterial fermentation in making yoghurt.
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In this Journey to Discovery, our story begins with a puzzling observation of strange sequences in the
DNA of bacteria, which were completely mysterious at the beginning and of interest to very few people.
But then a trail of clues began to emerge from several scientists that ultimately led to the discovery of
the natural function of CRISPR as an “immune system” that protects bacteria against viruses. This pro-
vided an answer to the problem that our company faced of understanding how certain bacterial cul-
tures survived phage attack. Eventually, those studies laid the foundation for today’s “CRISPR craze,”
the most disruptive new biotechnology to emerge in the past two decades.
Clue 1: CRISPR sequences in bacterial genomes

The first clue in the CRISPR story can be traced back to 1987. At that time, Ishino and colleagues
in Japan serendipitously stumbled upon an unusual DNA sequence composed of five identical
sequences of 29 nucleotides (Figure 3). They noted the sequence in one of their publications but
said that it had no known “sequence homology … and biological significance.” This observation
received little attention and was ignored and overlooked for many years.
The curious ~29 nucleotide repeats resurfaced a decade later in the late 1990s when the genomes
of many prokaryotes were being sequenced. Only a few people, however, were curious enough
about them to point them out. Francisco Mojica was particularly noteworthy among those individ-
uals, since he not only noticed them, but also chose to pursue them for his research. Mojica found
these sequences in more than 20 different types of bacteria. Since they were so prevalent, they
must be doing something important, but what?
Researchers needed a unifying name to refer to these mysterious sequences. In 2002, the field con-
verged upon the acronym CRISPR for Clustered Regularly Interspaced Short Palindromic Repeats.
CRISPR encompasses short stretches of repeated DNA that are clustered together at a particular
genetic locus and are partially palindromic: they read the same read backwards and forwards,
as with the words “mom” or “racecar.” These intriguing DNA repeats are typically separated by
seemingly random, at least at that time, DNA sequences named “spacers” (Figure 3, CRISPR
sequences). The name “Spacers” make these sequences sound boring, but they will emerge as
important players later on (see Clue 3).
Clue 2: CRISPR sequences are paired with cas genes

Genomes are composed of “coding” and “non-coding” regions. The coding regions are elements
of genes and are transcribed into RNAs, which in turn are translated into proteins (see Central
Dogma). Some of the non-coding regions in the genome are transcribed into RNAs that do not
make proteins but serve functional roles in the cell. We will see later that the CRISPR sequences
make such non-coding RNAs.
The genes are usually easier to spot in the enormously long sequence of bases that make up the
genome. Adjacent to the CRISPR region were certain characteristic genes, called CRISPR-associated
sequences (cas). One Cas gene that plays a particularly important role in this Narrative is called Cas9
(depicted in Figure 4). In bacterial genetics, the large majority of the DNA sequences are coding
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Figure 3. The CRISPR array in the genome of bacteria. Note that the repeats are identical in sequence
while each spacer is different. There can be as many as 50 such repeat-spacer elements.
sequences (over 90% of bacterial DNA typically consists of protein-coding sequences, whereas the
large majority of eukaryotic DNA is non-coding). One consequence of this more packed genome is
that several genes that encode proteins with related functions are often clustered together. Thus, the
co-occurrence and co-location of CRISPR arrays and cas genes strongly suggested that they are part
of a unified systems with a related biological function. Thus, elucidating the function of one could give
clues into the function of the other. But what was their biological function?
DNA sequences are most easily studied using computers, which can decipher information in long
strings of As, Ts, Cs and Gs (see Video 1 Whiteboard on Bioinformatics). For instance, are partic-
ular sequences coding (i.e., do they encode proteins per the Central Dogma of biology), or not? Are
similar sequences found in different organisms or are the sequences unique? Is the sequence of
As, Ts, Cs, and Gs random, or do they form some sort of a pattern?
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Figure 4. Cas genes are located next to the CRISPR array.
Video 1. Whiteboard on Bioinformatics
Eugene Koonin and Kira Makarova at NCBI (the National Center for Biotechnology Information,
within the NIH) were masters at deciphering patterns in DNA sequences. They attempted sev-
eral times to ascribe functions to the Cas proteins associated with CRISPR loci (failure is part of
science, and this is why multiple attempts at searching turn into “re-search”). Eventually, they
established that Cas proteins are related to other known proteins that interact with DNA and
RNA molecules. In particular, they had some distantly related features in common with proteins
involved in a defense system known as RNA interference that degrades the RNA of invading
viruses. This clue led them to hypothesize that the Cas proteins may be part of an ancient pro-
karyotic defense system, which we will see soon is correct. However, the CRISPR-Cas defense
system is different from RNA interference and indeed unlike anything that was previously known
or discovered.
Clue 3: CRISPR “Spacer” sequences show homology to phage DNA

What about the “spacer” sequences within the CRISPR array, the seemingly random sequences
between the CRISPR repeats? The most basic computational analysis is a homology search: does
a string of unknown or mysterious nucleotide sequences match something known and previously
characterized genetically or functionally. Such a match might provide a powerful clue as to the
function of the unknown sequence. Did the spacer sequences match any other sequences in DNA
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databases? Several scientists found matches, and they were surprising ones indeed. The spacers
showed homology to DNA sequences from bacteriophages and plasmids, invasive elements of
bacteria. This clue raised a new element of intrigue: why would there be viral sequences between
repeated bacterial DNA elements? The answer came together in the Discovery, which is featured
next.
The Discovery: CRISPR-Cas is a bacterial immune system

I was part of a team at Danisco (a DuPont company) working on Streptococcus thermophilus,
a bacterium globally formulated as a starter culture for the industrial fermentation of milk into
yoghurt and cheese. Back in the early 2000s, DNA sequencing was used to determine the complete
genomic sequence of many medically and industrially important bacteria. My first job at Danisco
was to sequence and assemble the genomes of bacteria used as probiotics and starter cultures
in food fermentations.
I remember encountering the CRISPR repeat sequences in the early 2000s when we were sequenc-
ing the complete genome of a popular commercial probiotic strain used in yoghurt fermentation
called Lactobacillus acidophilus NCFM. The repeating structure of the CRISPR locus was so mes-
merizing and genetically peculiar. We had no idea what they did, but because we encountered
them so frequently in genomic sequences, our instinct was that they must be doing something
important. Together with my Danisco colleagues, notably Philippe Horvath, we decided to launch
a research project aimed at solving the mystery of CRISPR.
As is often the case in science, we did not solve the CRISPR puzzle in one go, but we were able
to bootstrap our way to an eventual answer. We got there in two steps: the first was to analyze
CRISPR sequences from a wealth of genomic data that we had at our company, which led us to a
hypothesis. The second step involved performing experiments to test our hypothesis. In addition
to reading, you can also hear my description of the discovery in Video 2.
Video 2. The discovery: CRISPR functions as an immune system to protect bacteria
against viral infection. Rodolphe Barrangou describes his discovery that CRISPR
functions as an immune system to protect bacteria against viral infection.
As is often the case in science, we did not solve the CRISPR puzzle in one go
Step 1: Clues from Analyzing DNA Sequences of the CRISPR Locus

Rapid Evolution of the CRISPR Locus
Philippe and I quickly discovered that the CRISPR sequences were highly variable, even between
different strains of the same species (a strain is a subtype of a species with a distinct genetic
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background). This hypervariability of CRISPR spacer sequences proved useful, as we were able
to use them as characteristic identifiers of different strains of S. thermophilus. In fact, genetic
subtype screening is the basis for the very first CRISPR patent that was ever submitted, in 2004.
Eventually, we determined the CRISPR sequences of thousands of S. thermophilus strains isolated
across the globe and used for worldwide fermentation of milk into yoghurt and cheese. We now
had a wealth of DNA sequence data for the CRISPR locus at our disposal. Might these data reveal
the function of CRISPR?
During our CRISPR subtype analysis, we noticed something remarkable, which steered us on the right
track. Fortunately, Danisco kept excellent records of numerous S. thermophilus strains over decades.
Looking at a diverse collection of strains, we could group families of strains based on similar CRISPR
DNA sequences and cross-reference this information with their phage sensitivity. Remarkably, cer-
tain strains of S. thermophilus, grouped by us solely based upon the DNA sequence of their CRISPR
locus, were previously classified by Danisco as being particularly resistant to certain bacteriophages.
Conversely, strains with other CRISPR sequences were classified as being more phage sensitive.
Explorer’s Question: What might this result suggest to you?

Answer: CRISPR is a DNA sequence in search of a function. Remarkably, this result showed
a correlation between bacterial strains with similar spacer sequences in the CRISPR loci
and their sensitivities to particular phage. This result correlated a genotype (a particular
characteristic of the CRISPR DNA sequence) to a phenotype—whether the bacteria were
resistant or susceptible to phage attack. This is not proof that the two are connected but
provides a hint that they might be.
We also noticed a second surprising result: the CRISPR locus could change over time. Danisco not
only kept records of different subtypes of S. thermophilus but they also kept records of individual
strains over time. We had specific examples of a particular bacterial strain that was sampled and
sequenced several years apart and could compare the sequences. While the vast majority of the
genome remained unchanged, we noticed that the number of repeats in a CRISPR array could
expand within a given strain even over months (Figure 5). Evolution usually sculpts changes in the
genome through the relatively slow process of spontaneous mutations and natural selection (see
Narrative by Koshland on Mutations). Our results, however, suggested that the CRISPR locus was
evolving and changing much more rapidly than the rest of the bacterial genome. The CRISPR locus
was not a static genetic feature; rather, new repeats and spacers could be added on top of those
that existed in the ancestral strain. Where did these sequences come from?
The CRISPR Locus has Homology to Viral Sequences

At the same time and independently of Koonin and Mojica (see their work described in Clue 3), we
used bioinformatics to look for meaningful homologies of CRISPR sequences with other sequences
that had been deposited in DNA databases. Besides the obvious matches of CRISPR repeats
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Figure 5. The CRISPR locus in a bacterial strain can expand over time.
with one another, we consistently observed imperfect but believable matches between the spacer
sequences and eukaryotic viral sequences, such as monkeypox, hepatitis, influenza, and even HIV.
Why would there be eukaryotic viral sequences in the CRISPR locus? Was this contamination or
an artefact? We had little reason to believe that eukaryotic viruses were infecting bacteria. Still,
these homologies were not random “noise,” because the likelihood of only picking up homologies
with viral sequences from a large DNA database (filled mostly with prokaryotic and eukaryotic
sequences) was very low if it was a totally random result.
Still, the homology with eukaryotic viruses did not make sense. However, this initial finding indi-
cated to us that we should not solely focus on the repeats, the obvious DNA sequence elements that
actually defined “CRISPR.” Instead, we needed to pay attention to the spacer sequences between
the repeats, which at the time were thought to be less important or wholly unimportant. As Pasteur
once said, “chance favors the prepared mind,” and we were now ready to pay attention.
A Match between CRISPR “Spacers” DNA Sequences and Phage that Attack Them
As mentioned previously, bacteria, including those used in food manufacturing, are subject to
phage destruction. Accordingly, while we were sequencing S. thermophilus strains, the Danisco
team was also busily sequencing the genomes of the phages found in our manufacturing environ-
ments. We were fortunate to have both sets of sequence data available within our company. With
more pieces and clues available, the puzzle was starting to take shape.
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By sequencing CRISPR loci of our commercial bacterial strains and the viral sequences of the
phages that attacked them, we came across an extraordinary finding that was a turning point
in the project. The CRISPR spacers were sometimes perfect nucleotide matches to the phage
sequences that attacked them! The expansion of the CRISPR locus actually occurred by addition
of DNA sequences matching those of phages. Although circumstantial, and not evidence per se,
this was a rather compelling observation linking the CRISPR loci to phage infection.
we came across an extraordinary finding that was a turning point in the project.
The CRISPR spacers were sometimes perfect nucleotide matches to the phage
sequences that attacked them!
To build this connection further, we had the amazing fortune of having frozen stocks of historical
strains within the Danisco collection that had been isolated and characterized over decades. We
unearthed strain derivatives that had been exposed to industrial phages. In some of those cases,
we observed that the CRISPR loci had expanded through the addition of spacers with an exact
nucleotide match to sequences found in the genome of the infecting phage that plagued our
industrial plants. In some notable cases, we were able to observe larger CRISPR loci in phage-
resistant bacteria that had been generated by our company, sometimes decades ago. Thus, the
acquisition of a CRISPR spacer with a match to a particular phage was correlated with the ability
of that bacterial strain to resist death by the phage. The plot thickened.
When we realized this, we had a strong suspicion that CRISPR was linked to immunity. Immunity
refers to the ability of an organism to resist infection. Mammals, for example, have the ability to
Figure 6. A nucleotide

match between a CRISPR
spacer and the DNA of a
phage that previously infect-
ed the bacteria. A spacer
sequence in a bacterial strain
(colored) matched the DNA
sequence of a phage strain to
which it developed resistance.
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store a memory of a prior attack by a pathogen and then respond swiftly to a subsequent attack
and defeat the invader. This ability to remember and respond with a counter-attack is called
“adaptive immunity,” which is mediated by specialized white blood cells called T cells and B cells.
Adaptive immunity is the basis of vaccination, which involves an intentional exposure to an inac-
tivated pathogen to create a stored memory for a possible future infection. Based upon our clues
that (1) CRISPR loci evolved over time, and (2) that CRISPR spacers seemed derived from phage
genomes, we hypothesized that CRISPR served as an adaptive immune system for bacteria and
constituted a genetic vaccination record of infections.
Step 2: Experiments to Test Whether CRISPR Serves an Adaptive Immune System

After the clues came together, our next step was to devise experiments that would enable us to test
the link between CRISPR loci and phage resistance. Of course, as scientists working in industry, we
first filed for a patent application (time-stamped August 2005). Once a patent is filed, inventors have
12 months to reduce their invention to practice. Not only were we tackling a challenging problem,
but we were racing against this 1-year clock to convert the patent with evidence obtained from
experiments.
We developed three sets of experiments to demonstrate the connection between CRISPR and
resistance to phage infection.
CRISPR loci acquire phage sequences

In the first series of experiments, we aimed to test the hypothesis that the CRISPR locus expands
after the phage exposure and accompanies development of resistance. We exposed a S. thermo-
philus strain to a phage and let nature run its course. Experimentally, this was done by exposing a
bacterial broth culture to a solution of phages and then distributing it on an agar plate (Figure 7).
Expectedly, the majority of the bacterial population succumbed to the phage and died. However,
an occasional robust bacterium could build resistance against phage; this small proportion of the
bacterial population (a couple in a million) grew despite the presence of the virus.
Resistance can arise by spontaneous mutations in the bacterial genome; for example, in a bacterial
protein to which the phage needs to attach in order to inject its genome into the bacteria. Indeed,
resistance through spontaneous mutations was the basis of the Nobel Prize winning experiment
by Salvador Luria and Max Delbruck, which is described in the Narrative on Mutations by Koshland.
However, in addition to spontaneous mutations, we hypothesized that some phage may become
resistant through CRISPR immunity. (For discussion of various strategies of how bacteria can
become resistant to phage, see Dig Deeper 1.)
In our experiment, we selected phage-resistant variants (colonies of bacteria that survived on

the plate), amplified the DNA in their CRISPR loci by a method called polymerase chain reaction
(PCR) and sequenced the DNA (Figure 8). Generally, the bacteria became resistant to one phage
strain to which they were exposed but did not become resistant to another closely related phage
that they had not seen. On the bar graph below, a value of 1 indicates a maximal “phage kill” of
19
Figure 7. Phage attack of the bacterium Streptococcus thermophilus and the identification of
resistant mutants. When placed on agar plates, colonies of resistant bacteria grow up from an
individual resistant bacterium through many rounds of cell division.
the original bacterial strain; smaller numbers indicate less-effective killing by the phage (note that
this is logarithmic plot that decreases by powers of ten). Thus, as shown in Figure 8, this bacterial
colony that was exposed to a phage A became resistant to phage A but not phage B. This was a
heritable trait that the original resistant bacteria passed on to their progeny.
Explorer’s Question: This bacteria colony acquired two spacers. A comparison of the bac-
terial spacer sequences to phage A and B genomic sequences is shown above. What do
you conclude about the nucleotide specificity required for phage immunity?
Answer: Spacer 1 is an exact nucleotide base match to phage A, but a poor match to phage
B. Spacer 2 has a single nucleotide mismatch with both phage A and B. From these data
and the information that this bacterium is not resistant to phage B, you might conclude
that spacer has to be an exact match to a phage sequence in order to confer immunity.
In addition to this one experiment, you would want to see this finding substantiated with
more examples. Additional results have shown that some mismatches can be tolerated at
the distal end only.
20
Figure 8. Bacteria exposed to phage A become selectively resistant to phage A and acquire new
spacers with sequence matches to phage A. The phage A-resistant bacteria were tested for killing
by phage A and B. A value of “1” indicates a similar kill frequency to naïve bacteria that were not
exposed to either phage. Lower values (note: this is a logarithmic plot) indicate resistance. Note that
the bacterial strain became resistant to phage A was not resistant to phage B. DNA sequencing of
the CRISPR locus showed that this strain acquired two new spacers, both of which had a perfect
match to DNA sequences in phage A but not in phage B.
21
We also performed the converse experiment of infecting bacteria with phage B and then looked
at the spacer sequence of the bacteria that developed resistance. In the result shown below
(Figure 9), a new spacer was acquired by the bacteria that showed an exact match to phage B
DNA but not phage A. And those bacteria were selectively resistant to phage B.
Figure 9. Bacteria exposed to phage B become selectively resistant to phage B and acquire a
new spacer with a sequence match to phage B. The phage B-resistant bacteria were tested for
killing by phage A and B. A value of “1” indicates a similar kill frequency to naïve bacteria that were
not exposed to either phage. Lower values (note: this is a logarithmic plot) indicate resistance. Note
that the bacterial strain became resistant to phage B was not resistant to phage A. DNA sequencing of
the CRISPR locus showed that this strain acquired a new spacer with a perfect match to DNA sequences
in phage B but not in phage A.
22
Figure 10. Bacteria exposed to phage A and B become selectively resistant to both phage and
acquire a new spacer. Bacteria exposed to a cocktail of both phage A and B were tested for killing
by phage A and B. A value of “1” indicates a similar kill frequency to naïve bacteria that were not exposed
to either phage. Lower values (note: this is a logarithmic plot) indicate resistance. Note that the bacterial
strain became resistant to both phage A and phage B. DNA sequencing of the CRISPR locus showed that
this strain acquired two new spacers with perfect matches to DNA sequences shared by phage A and B.
We also repeated the experiment with a phage cocktail of phage A and B (Figure 11). In this case,
bacteria acquired immunity to both phage. The bacterial colony that we selected acquired two
spacers that were perfect matches to both phage strains (strains A and B are very similar and
23
Figure 11. Swapping spacers by genetic engineering switches phage resistance. A bacterial
strain resistant to phage strain B (see Figure 9) had its S4 spacer removed and replaced with the
spacers (S1, S2) associated with resistance to phage A. After this swap, the genetically engineered
bacteria lost resistance to phage B and gained resistance to phage A. A value of “1” indicates a similar
kill frequency to naïve bacteria that were not exposed to either phage. Lower values (note: this is a
logarithmic plot) indicate resistance.
Explorer’s Question: The above resistant bacteria acquired two new spacers. What do
you think would happen if one of the two spacers was deleted?
Answer: Each spacer shows sequence identity to both phages. So one spacer should be
sufficient to confer resistance to both phages. However, having two spacers might make
resistance more efficient.
share regions of identical sequences as well as other regions with minor differences, as in the
examples shown above).
In summary, the experiments we performed showed that bacteria that survive exposure to phage
acquire novel CRISPR spacer sequences that match the antagonistic viral sequences.
24
Explorer’s Question: Are the above data sufficient to validate the hypothesis that the
CRISPR locus provides immunity to phage?
Answer: These data SUGGEST that there may be a link between the CRISPR locus (the
CRISPR genotype) and phage resistance (the antiviral phenotype). The experiment DOES
NOT PROVE the link. It shows that phage infection leads to resistance AND acquisition
of a new, matching spacer, but not that phage infection leads to resistance BECAUSE
of the acquisition of the spacer. While we had an exciting scientific piece of data in the
story, we still had to demonstrate the direct causal link between CRISPR content and viral
resistance.
CRISPR spacers confer phage resistance

In the second series of experiments, we genetically altered the CRISPR spacer content our-
selves by genetic engineering, without phage infection. We then could ask the question: if we
introduced a new CRISPR spacer corresponding to a particular phage, would that genetically
engineered bacterium become “immune” to that phage? This experiment would test whether
there is direct link between the CRISPR spacer content and the sensitivity of the strain to
viruses.
Conveniently, during in my Ph.D. studies, I learned how to alter the genetic content of lactic acid
bacteria (like Streptococcus thermophilus), and had access to the required genetic engineering
tools, which were not readily available at the time. Thus, I was rapidly able to engineer the CRISPR
locus by adding, removing, or swapping “spacer” regions. We showed that by:
A) adding a spacer: phage resistance was acquired;

B) removing a spacer: resistance was lost;
C) swapping CRISPR content between two strains: their respective phage resistance was
switched.
Explorer’s Question: Which of the above experiments would be considered a “loss of func-
tion” and which a “gain of function”?
Answer: Experiment B is a “deletion” that would result in a “loss-of-function.” Experiments
A and C are “gain of function” experiments. In both cases, the genetic engineering results
in the acquisition of a new phenotype of function: resistance to phage.
An example of the data from experiment C is shown below. In this case, we took the bacterium
that acquired resistance to phage B and swapped out its spacers for the ones that were associ-
ated with resistance to phage A. When we did the spacer swap and retested for phage sensitivity,
indeed we found that phage resistance was now switched to phage A (Figure 11).
25
Cas genes are involved in acquisition and interference

As previously mentioned in Clue 2, CRISPR loci are typically in close proximity to cas genes, and
genetic association typically correlates with functional association. Given the association, we next
set out to determine whether cas genes were involved in phage resistance. We used a molecular
biology technique called a “knock out,” which inactivates a gene by inserting a piece of DNA that
disrupts the coding sequence, thereby precluding the translation of a functional protein.
We found that the two largest cas genes in S. thermophilus, cas9 and csn2, are essential for
CRISPR function, but for different reasons. By inactivating csn2, the CRISPR locus was unable to
acquire new spacers from phage, implicating this gene in the vaccination process. By inactivating
cas9, CRISPR sequences were acquired but the bacteria were unable to resist phage attack, impli-
cating this gene in the immunity process (Figure 12). These results show that different cas genes
Figure 12. Inactivation of the Cas9 gene disables phage immunity. A piece of DNA was inserted
into the middle of the Cas9 gene, which disrupts the production of a functional Cas9 protein. After this
“knockout” of Cas9, resistance to phage infection is lost. A value of “1” indicates a similar kill frequency
to naïve bacteria that were not exposed to either phage. Lower values (note: this is a logarithmic plot)
indicate resistance.
26
are involved in spacer acquisition and subsequent attack on phage invaders. The largest gene,
cas9, would encode what turned out to be the most revolutionary molecular tool in a generation,
as we will discuss later. While the involvement of CRISPR spacers in phage resistance had been
posited by others, their reliance on associated cas proteins was unknown at the time.
What happened next?

We were quite excited about these results. We thought that we had definitive proof of a sophis-
ticated adaptive immune system in bacteria. We used our three experiments described above to
support our patent application. However, it took Philippe and myself 6 months to convince the
management at Danisco that our results were worth publishing. It also was not trivial to convince
Science magazine and its reviewers to have interest in publishing a study on a peculiar genetic
locus from a yoghurt starter culture. But eventually, the paper was submitted in 2006 and pub-
lished in 2007 (see Dig Deeper 2 for more information on this story of publishing the work).
While our study showed that CRISPR-Cas functions as an adaptive immune system, it did not
explain how it works. In 2008, considerable progress was made when two groups characterized
the mechanism of action of the CRISPR-Cas immune system. First, van der Oost and colleagues
showed that CRISPR immunity is driven by RNAs (called crRNAs) that are transcribed from the
CRISPR locus; each contains one spacer, thus defining a particular earlier invader of that bac-
terial strain. This established that CRISPR immunity is RNA mediated. Second, Marraffini and
Sontheimer showed that the phage DNA is the target of CRISPR interference. After relatively slow
progress since the first observation in 1987, in a relatively short timeframe it was established that
CRISPR is a DNA-encoded, RNA-mediated, DNA-targeting immune system.
Attention also turned to Cas9 and the mechanism of this important enzyme came into focus.
We, in collaboration with the Moineau laboratory in Canada, showed in 2010 that Cas9 is an
endonuclease that cleaves plasmid and phage DNA during infection. Equipped with this scientific
understanding of the CRISPR-Cas molecular machinery, the Doudna and Charpentier laborato-
ries repurposed Cas9 to become a versatile genome cleavage tool in vitro in 2012 (see CRISPR-
Cas9 Key Experiment by Doudna et al.). The following year, multiple labs concurrently showed in
human and bacterial cells that Cas9 could be programmed to selectively target DNA sequences
of interest and drive genome editing by the endogenous DNA repair machinery. Although multi-
ple groups were performing similar studies concurrently, teams lead by Feng Zhang and George
Church would be the first to publish back-to-back papers in Science early in 2013, showing that
Cas9 nucleases could generate efficient genome editing in human cells. This revolutionized the
genome editing field and triggered the CRISPR craze that has taken hold ever-since.
Shortly after the initial demonstration that CRISPR enabled genome editing, Cas9-based editing
was extended to many different cells and many entire living organisms, resulting in perhaps the
most explosive new biotechnology tool to come along since DNA cloning and DNA sequencing.
With DNA sequencing tools, scientists could “read” the genetic blueprint (the genome) of any
organism. With CRISPR-Cas, scientists now have a tool to “rewrite” the genome. We will explore
some applications of this new technology in the Frontiers section.
27
With CRISPR-Cas, scientists now have a tool to “rewrite” the genome

The growth of the CRISPR field has been amazing to witness. In 2008, we held the first scientific con-
ference on CRISPR-Cas systems at UC Berkeley (organized by Jill Banfield and myself). The meeting
was small, because only about two dozen individuals in the whole world had any interest in these
systems. There were relatively few papers published on CRISPR in the scientific literature. However,
the meeting started a series of field-changing collaborative efforts, and life-altering exposures to
CRISPR for investigators, students, and post docs, many of whom are now world-famous scientists.
Now, there are hundreds of thousands of scientists using CRISPR-based technologies in academia
and industry and thousands of papers involving CRISPR being published every year. Starting from
humble beginnings – curiosity about patterns of nucleotides in the bacterial genome – CRISPR is now
revolutionizing all of biology.
Starting from humble beginnings – curiosity about patterns of nucleotides in the

bacterial genome – CRISPR is now revolutionizing all of biology
Part II: Knowledge Overview

Bacteria occur broadly in nature and are nearly ubiquitously present in various habitats and environ-
mental niches on the planet. Recent microbiome studies have unearthed amazing bacterial diversity
in mixed microbial populations in water, soil, plants, animals, and humans. Bacteria are also widely
used in the manufacturing of fermented food and have been domesticated for food preservation
processes for millennia. Likewise, the viruses that infect bacteria, called bacteriophage or phage,
have now been studied for over one hundred years and have been detected throughout the globe.
Bacteriophage are believed to be the most abundant and the most diverse biological entity on the
planet. Yet bacteria thrive, despite the ubiquitous presence of predatory viruses. How these two bio-
logical enemies, phages and their bacterial host, engage in a sustainable arms race is a fascinating
puzzle. In the first part of the Knowledge Overview, I will describe our current view of how bacteria
use the CRISPR-Cas9 immune system to defend themselves against phage at the level of “genomic
warfare” (see also Video 3). In the second part, I will describe how this system has been re-purposed
for genome engineering.
Video 3. A summary of how CRISP immunity works by Rodolphe Barrangou.
How these two biological enemies, phages and their bacterial host, engage in a
sustainable arms race is a fascinating puzzle
28
How CRISPR immunity works

Generally, CRISPR-Cas systems are composed of a CRISPR array associated with cas genes (see
Journey to Discovery). The CRISPR array consists of a series of conserved CRISPR repeats inter-
spersed with spacers, sequences from invasive genetic elements such as bacteriophages and
plasmids that the bacteria acquired over time to constitute a genetic “vaccination card.” If the
bacteria encounter foreign DNA that matches the sequence of one of their spacers, for example,
they are attacked by a phage that they have encountered previously, the spacer will direct one of
the Cas proteins to cut the foreign DNA and halt the attack.
Bacteria have evolved many different flavors of CRISPR-based defense systems, reflecting the
diversity of both bacteria and the viruses that prey upon them, and their widespread occurrence
and distribution reflects the nearly ubiquitous presence of viruses on our planet. Discussion of
other CRISPR-Cas systems can be found in Dig Deeper 3. Here, I will focus on the CRISPR system
that has been most widely repurposed for genome engineering, from the bacteria Streptococcus,
for which the Cas protein that cuts the foreign DNA is called Cas9.
The three steps of CRISPR immunity are discussed below: (1) acquisition, (2) expression, and
(3) interference.
Acquisition
In this first step, a segment of the invasive DNA is copied and fused to a new CRISPR repeat at the ter-
minal end of the CRISPR array by a series of bacterial enzymes (Figure 13). This allows unidirectional
addition of novel spacers iteratively over time. The series of spacers captured in the CRISPR array thus
constitutes a historical genetic record of the infection. By capturing a piece of the invasive DNA into
the CRISPR locus, this dynamic system is adaptive (evolution in response to an environmental stimulus,
such as a viral attack). It is also heritable, since once integrated into the bacterial DNA, the incorporated
viral sequence will be subsequently passed on to the next generation of bacteria once the cell divides.
Vaccination is not only effective against the original virus but also any related variant which contains
the captured sequence, providing opportunities to build immunity against families of viruses when the
acquired sequence is conserved across a group of phages.
Expression
In the expression process, the CRISPR array is transcribed into a precursor CRISPR RNA molecule
(pre-crRNA) (Figure 14). The pre-crRNA is then cleaved and processed into a series of small
CRISPR RNAs (crRNAs) by cleavage within the CRISPR repeat sequence. This cleavage reaction
involves another RNA, called tracrRNA, and the Cas9 enzyme. Each crRNA that is produced con-
tains the sequence derived from one spacer. The spacer sequence acts as a “search” query to
detect potential invasive elements that match prior infection events and allows the host to trigger
the immune response when a match is found.
Since viral infections can take over the host to promote virus replication within minutes, the cell
must respond very rapidly to the infection if it is to survive. Therefore, CRISPR spacers and Cas
29
Figure 13. Acquisition of new CRISPR spacers. After invasion of a bacteriophage, some of the phage
genome is cleaved and this DNA (now called a “spacer”) is inserted at the leading end of the CRISPR
array in the bacterial genome.
proteins are always expressed so that the cell can mount a timely immune response. However, the
transcription of the CRISPR components can also be elevated after a viral attack, which sounds
the alarm and generates a more robust response.
Interference
In the final step that targets the phage genome, a crRNA and a tracrRNA form a complex with the
protein Cas9. The fully loaded Cas9 then scans along DNA in a random search to find a sequence with
a perfect match to the crRNA along with another more general recognition cue called a PAM sequence
(see Dig Deeper 3). Cas9 scans along the DNA through a random process known as diffusion, which
is discussed in the Narrative by Prakash. If a perfect sequence match to the crRNA sequence is found,
then the Cas9 stops scanning and triggers an endonuclease activity; endo means “internal” (i.e. within
a long DNA molecule) and nuclease is an enzymatic activity acting to sever a nucleic acid. In the case of
Cas9, the enzyme cuts across both strands of the DNA helix, breaking a single long molecule into two
parts (Figure 15). This single cutting activity is sufficient to foil the replication plans of a phage, which
needs to preserve and propagate its genome as a single intact DNA molecule. This enables this bacte-
rial immune system to search for sequences that match its spacer vaccination record and specifically
destroy genetic elements that contain that genetic signature.
The mechanism for Cas9 targeting described above raises an interesting question. Why doesn’t
CRISPR-Cas9 target the CRISPR array in the bacterial genome, because this DNA is also a per-
fect match for its crRNAs? The answer is that Cas9 requires a second nucleotide cue in addition
30
Figure 14. Expression and cleavage of CRISPR RNAs. Initially, a long RNA is produced from the
CRISPR locus, which is then cleaved into individual spacer-repeat units by Cas9.
to the sequence match to the crRNA. This cue is a short sequence signature (such as NGG, where
N = any nucleotide) next to the spacer sequence, which is called the protospacer adjacent motif
(PAM). Cas9 must first bind the PAM in the DNA, and only then can it interrogate the flanking DNA
for a match for its crRNA. If there is a complete sequence match, then the Cas9 endonuclease is
activated and cuts DNA. Importantly, the reliance on PAM target recognition prevents self-targeting
of a bacterium’s own CRISPR array, because the spacers in the CRISPR array are flanked by the
repeats, which never possess PAM sequences. This allows bacteria to distinguish self versus non-
self, a key part of any immune system, and thus prevent an autoimmune response.
Repurposing Cas molecular machines for genome editing

Genome editing involves rewriting the DNA sequence at a very specific location. For example, in
the human genome, one may want to alter a single nucleotide out of three billion, for example, to
correct a single base pair mutation that produces sickle cell anemia (see Narrative on the Laws of
Inheritance by Tilghman). The overall strategy for making an edit is to first cut the DNA close to
where you want to rewrite the sequence, and then introduce a new sequence during the natural
process of repairing the DNA, as will be described in this section.
The trick is how to cut the DNA at a defined location. There were methods of cutting genomic DNA at spe-
cific locations before CRISPR. However, they involved engineering proteins to recognize and cut specific
sequences. Designing and producing these specialized proteins is very slow and difficult to implement.
31
Figure 15. The interfer-

ence step in which Cas9
recognizes and cuts the
phage genome. The rec-
ognition step by Cas9 first
involves finding a simple
nucleotide sequence (often
just three nucleotides; the
protospacer adjacent motif
or PAM). Cas9 then partial-
ly unwinds the target DNA
and probes for a sequence
match (angled lines)
between the target DNA
and the crRNA. If there is a
sequence match, a crRNA-
DNA duplex is formed,
which then activates the
Cas9 nuclease to generate
a double-strand break in
the target DNA.
32
In contrast, designing and producing specific DNA or RNA sequences is a routine process in laboratories
around the world. Therefore, the world of genome editing changed in 2012 when Doudna, Charpentier and
colleagues showed that the CRISPR machinery involved in the bacterial adaptive immune system could be
re-engineered into a simple and programmable tool to cut DNA at virtually any defined sequence with only
DNA engineering. Cas9 could be used as is; no protein engineering was necessary. They also found that
the two key RNAs (tracrRNA and crRNA) involved in Cas9 cutting could be fused into a “single-guide” RNA
(often abbreviated sgRNA) (see the Key Experiment by Doudna) (Figure 16), simplifying the system further.
Their biochemical experiment (performed outside of cell in a test tube environment), raised the possibility
that expressing Cas9 and a sgRNA in a cell would result in a specific DNA cut in a genome. Shortly thereaf-
ter, Feng Zhang, George Church, and others showed that this strategy works in a variety of cells and began
generating tools to make this strategy for genome editing in cells broadly accessible.
The trick is how to cut the DNA at a defined location

Typically, DNA breaks are toxic and can even be lethal, especially when a cell tries to replicate its
genome and divide. DNA cleavage can occur as a result of environmental insults such as radiation as
well as other causes (see Narrative on Mutations by Koshland). To protect itself against such damage,
cells have ways of repairing broken DNA. Once the DNA is cut, the two cleaved DNA ends can be
rejoined in a process known as DNA repair (Figure 17). DNA repair is an important and complex pro-
cess (see iBiology video for a more in-depth explanation), and here I will only outline the basics and
particularly how it relates to DNA editing. In most organisms, there are two distinct pathways that
repair double-stranded DNA breaks: non-homologous end joining (NHEJ) and homology-directed
repair (HDR). NHEJ unpredictably adds a few nucleotides or generates a single base mutation, yielding
a genetic abnormality at the site of cleavage and repair, analogous to the way in which repair of a cut
on your skin leaves a scar. If the scar is in a non-essential, non-coding region of the genome, this quick
but imprecise repair can be tolerated. But in a coding region of a gene, this can be disruptive. For example,
the addition or deletion of nucleotides in numbers other than multiples of 3 would change the reading
frame and disrupt the coding sequence, yielding a faulty series of amino acids and/or prematurely
terminating translation and producing a truncated protein. Although such inaccuracies introduced by
CRISPR-Cas9 cutting plus NHEJ repair can be deleterious for the cell, they can be very useful for scientists,
by allowing researchers to study the consequences when the function of a particular gene is permanently
inactivated (referred to as “knocking out” a gene or creating a knockout).
Homology-directed repair (HDR) (Figure 17), on the other hand, produces error-free repair natu-
rally or can be deployed by scientists to introduce a well-defined edit. HDR makes use of a DNA
template with a similar sequence (a similar nucleotide sequence is called a homologous sequence)
to the one that was cut in order to execute the repair. Since most organisms are diploid, the nat-
ural repair template could be the second copy of the gene or potentially another similar sequence
found in the genome. However, scientists can fool the repair machinery by introducing a geneti-
cally engineered repair template that is very similar to the cut gene, but also contains the scien-
tists’ desired modification. By introducing many copies of this engineered template, it can compete
with the natural template. The key for HDR is that the repair template must contain regions that
are similar to the parts of the gene that flank the cut; these are called homology arms (Figure 17).
33
Figure 16. Joining of tracrRNA and crRNA into a single-guide RNA. See the story behind this design
and why it was important for genome engineering in the Key Experiment on CRISPR-Cas9 by Doudna.
34
Figure 17. Two processes for repairing a double-strand DNA break: non-homologous end joining
(NHEJ), and homology-directed repair (HDR). In the majority of cases, a cell will respond to a double-
strand break by introducing a few random nucleotides to seal the break (NHEJ), which can disrupt the
gene’s function. Alternatively, the cell can use a sequence in the genome that is highly similar to the
damaged sequence as a template to repair itself (HDR). A researcher can hijack this repair strategy
by introducing many copies of DNA that is highly similar to the damaged sequence, but also contain a
stretch of new sequence or a modification. During the repair process, this modified sequence can get
precisely incorporated into the gene.
35
These homology arms allow the template to bridge across the cut, and the damaged DNA can use
the template to repair itself, introducing the engineered modification in the process. This modifica-
tion can be a specific insertion, deletion, or replacement of the original sequence. The size of the
edit also can be variable, from a single base pair change to a deletion or insertion of many thousands
of nucleotides.
In practice, genome editing works as follows (see Figure 18):
1) F
irst, an editing point needs to be determined by bioinformatics inspection of the gene
sequence. One must find a 20-nucleotide editing site in your gene of interest with an adjacent
PAM site on the 3¢ end; the sequence should be unique and not found elsewhere in the genome.
This sequence will be used to design a complementary sgRNA.
Figure 18. Strategy of preparing DNA reagents for a genome editing experiment. Performing a gene
editing experiment usually involves introducing two pieces of DNA into the cell you want to modify:
1) a plasmid that expresses the Cas9 and a single guide RNA that is complementary to, and specific for,
a site in your gene of interest, and 2) a template (here, carried on a plasmid) that the cell will use to
repair the break and introduce your desired modification. This repair template contains DNA sequences
that match the DNA sequence around the cut site (called homology arms), so that the template is a
good substrate for HDR.
36
2) If using HDR to generate precise edits (rather than NHEJ to generate a knockout ), design
a repair template with homology arms adjacent to the Cas9 cut site and a central region
designed to introduce an edit during HDR. Examples of edits include:
- Insert a stop codon to generate a truncated protein
- Insert a frameshift to generate a knock out
- Insert a sequence of interest (add an amino acid, a motif, or a whole gene). Green fluorescent
protein (see Key Experiment on GFP by Chalfie) is now frequently added to - genes by this
method.
- Delete a sequence of interest (remove an amino acid, a motif, or a whole gene)
- Replace a sequence of interest (change an amino acid, a motif, or a whole gene)
3) Deliver two plasmids into your cells of interest: one plasmid that will express Cas9 and the sgRNA
designed in step 1 and another plasmid containing the repair template. (There are variations on this
theme, but all involve delivering Cas9 protein, a sgRNA and a repair template if editing by HDR).
In summary, CRISPR-Cas9 allows the re-writing of the genome, and scientists have been successful in
implementing this simple protocol in a wide range of organisms. With more effort and optimization,
it may prove to be possible to edit the genome of any life form on this planet.
As wonderful as CRISPR-Cas9 genome editing is, it does not work with 100% efficiency (often more like
5–50%). Therefore, some cells receive the correct edit, while others do not. Therefore, scientists need
some way to identify the cells with the correct edit (one way is to sequence the DNA at the editing site)
and then isolate those cells and grow them up. Finally, there is some concern about whether CRISPR-
Cas9 also cuts regions of the genome other than the intended cut site (“off-target” cuts), which can
generate mutations through NHEJ. Such mutations would be of particular concern for medical applications.
These off-target mutations can only be found by sequencing the entire genome. Understanding and
minimizing these off-target effects is currently an important area of research.
Other Uses of CRISPR-Cas9

A version of Cas9 has been engineered to lack nuclease activity, called dCas9 in which the “d” stands
for catalytically “dead”, or “deactivated”. Thus, dCas9 can be directed by an sgRNA to bind specific
DNA sequences, but not cut the underlying DNA. By fusing dCas9 to other proteins, a researcher can
precisely deliver these proteins to specific segments of DNA. In this way, dCas9 fusions can be used to
repress gene transcription, or activate gene transcription at specific sites in the genome. For a description
of these methods, see the Key Experiment on CRISPR-Cas9 by Doudna.
Part III: Frontier

CRISPR-based molecular machines are powerful genome editing technologies that
enable flexible alteration of genomes and transcriptomes.
CRISPR-Cas9 editing allows scientists around the world to re-write virtually any sequence in
genomes of species across the tree of life. The method is efficient, relatively easy and relatively
37
inexpensive, democratizing the leading edge of biology research. It is accessible to novices; many
undergraduates are doing genome editing in their summer research projects. It is transforming how
biological research is conducted, shaping the future of medicine, launching new companies, and
eliciting a debate of what should or should not be edited (see Closing Thoughts). This is not just a
new ripple in biotechnology—it is a tidal wave and we still don’t know how tall it will become.
The method is efficient, relatively easy and relatively inexpensive, democratizing

the leading edge of biology research
The relative ease of implementation of genome editing in animals and plants has already led to
many proof-of-concept studies that illustrate the therapeutic and agricultural potential of genome
editing, a small subset of which are shown in Figure 19. In this section, I will focus primarily on
applications of CRISPR-Cas9 genome editing for treating hereditary human diseases.
There are more than 6,000 genetic diseases documented to date for which a specific causal muta-
tion in the genome has been identified. For instance, consider sickle cell disease. In this case, there
is one “bad” letter (an A nucleotide is changed to a T nucleotide in sickle cells patients) in the gene
encoding hemoglobin, the protein in red blood cells that carries oxygen. The altered protein also
changes the shape of the cell, creating the characteristic sickle shape. Correcting the gene in the
right cell type in patients might cure the disease. If the gene were corrected in an embryo, then the
individual would never experience sickle cell disease in their lifetime. If the gene were corrected in
the germline (egg or sperm), then that correction would be passed on permanently and become
part of the gene pool.
CRISPR-Cas9 and the treatment of Duchenne Muscular Dystrophy

Let’s walk through one example of how CRISRP-Cas9 is being developed for the treatment of Duchenne
Muscular Dystrophy (DMD). DMD is characterized by muscle degeneration starting in childhood and life
expectancy in the teens, although some patients can live to adulthood with optimal medical care. DMD
is a recessive genetic disorder caused by a mutation in a gene that lies on the X chromosome. Women
have two X chromosomes. Thus, with a good copy of the DMD gene and one bad copy, they do not
develop DMD and may be unaware that they are carriers of the disease. Having one X chromosome
and one Y chromosome, the male offspring of a female carrier are less fortunate. If a boy inherits one
bad copy of the DMD gene from mom, he will get the disease, and approximately ~1/5,000 boys are
afflicted by this disease.
The genetic defect for DMD lies in a gene called dystrophin. Dystrophin plays an important role in
muscle health; when dysfunctional, muscles that operate organs such as the heart and lungs fail
to perform, leading to organ failure (typically heart or lung) and early death. The dystrophin gene
is extraordinarily large, composed of >3,500 amino acids. In eukaryotes, most genes are divided
into exons (coding amino acids) and introns (non-coding regions that are removed by splicing; see
Video 5 to learn about RNA splicing). Dystrophin has 79 exons, one of which has a faulty nucle-
otide that prematurely terminates the protein. The nature of the defect in the DMD gene and the
strategy to correct it is described in Dig Deeper 4.
38
Figure 19. Examples of CRISPR-Cas9 genome editing applications.
39
Video 5. Whiteboard video on RNA Splicing.
To study human disease, scientists often create a genetic defect in an animal model that is equiv-
alent to that found in humans. In the case of DMD, scientists have introduced the DMD mutations
into mice by genetic engineering. A naturally occurring, spontaneous DMD mutation was also identified
in dogs (now maintained in beagles). This dog model, in comparison to mice, shows clinical and
pathological features that are similar to the human disease.
To study human disease, scientists often create a genetic defect in an animal

model that is equivalent to that found in humans
How does one change the defective gene in an animal? Here, I discuss work using CRISPR-Cas9
repair of the dog model of DMD (see the Guided Paper by Amoasii et al. in the Reference list). As we
learned in the Knowledge Overview, gene correction involves delivering Cas9 and a sgRNA. To do
this, scientists chose a virus for the delivery, in particular a virus called adeno-associated virus
or AAV. These viruses were selected because they preferentially enter muscle cells. The genome
of this virus can be engineered to include the genome editing components. For further safety,
a muscle-specific promoter was used so that the Cas9 gene is expressed only in muscle cells.
The therapeutic virus is grown to large quantities in a laboratory and delivered to the DMD animal
by intravenous infusion or direct injection into the leg muscle tissue (Figure 20).
After delivering the AAV therapeutic virus and waiting 6–8 weeks, the scientists first tested whether
their CRISPR-Cas9 gene editing strategy corrected the genetic defect. Not all genes were corrected,
as revealed by gene sequencing. However, muscle cells are somewhat unique in containing multiple
nuclei; thus, one might not need to correct all of the genes to create a functional muscle cell; perhaps
one gene per muscle cell might be enough. Furthermore, some studies indicate that even the recovery of
>15% of functional dystrophin could have clinical benefit for patients. Therefore, an important parameter
to measure was the restoration of dystrophin protein in the DMD dogs after CRISPR-Cas9 therapy.
This was tested using a technique called immunofluorescence, in which an antibody is used to probe
for the presence of the dystrophin protein. In the DMD dog, the gene defect results in lack of production
of dystrophin and immunofluorescence staining of muscle looks dark (Figure 21). However, when the
gene is corrected by CRISPR-Cas9, then the bright staining indicates that the normal protein is now
produced. The level of dystrophin recovery varied in different muscles but many muscles were restored
to >50% (including >90% in the heart).
Importantly, the abnormal pathology of the muscle in the DMD dogs was restored to a healthier
state by CRISPR-Cas9 therapy (Figure 22).
These results provide a promising basis for the development of engineered viruses carrying CRISPR
payloads to treat patients with DMD. More generally, this study illustrates how CRISPR-based
40
Figure 20. Steps in genome editing of a DMD animal model. See Dig Deeper 4 for more details
on the repair strategy.
genome editing approaches can be exploited to target specific mutations associated with disease
by co-opting the endogenous DNA repair machinery to correct a specific genetic defect. Many com-
panies are working to develop CRISPR-Cas9 therapies to treat many human diseases. Stay tuned and
follow the news. The first human clinical trials are happening as this Narrative was being written. This
is going to be an exciting area with broad implications for human health.
Other examples of uses of CRISPR-Cas technology

Within a couple of years of the genesis of CRISPR technology, hundreds of laboratories in industry
and academia have been performing genome editing of commercial plants such as corn, soybean,
wheat, rice, and sunflower, as well as vegetables and fruits including mushrooms, tomatoes, lettuce,
strawberries, apples, and other crops (tobacco, sweet potato, cassava). As an example, using
CRISPR to knock-out the gene responsible for browning in the white button mushroom, scientists
have extended the shelf-life of produce. Other obvious goals include targeted genetics to increase
41
Figure 21. Evidence that a defective dystrophin gene is corrected by CRISPR-Cas9 in a dog model
for DMD. Derived from Amoasii, L. Gene editing restores dystrophin expression in a canine model of
Duchenne muscular dystrophy. Science 2018;362:86–91. Copyright reserved by Science.
Figure 22. CRISPR-Cas9 therapy corrects muscle pathology in the DMD dog model. Derived from
Amoasii, L. Gene editing restores dystrophin expression in a canine model of Duchenne muscular
dystrophy. Science 2018;362:86–91. Copyright reserved by Science.
yield, drought tolerance, water usage, pest resistance, and optimizing lipid and protein composition
for increased nutrition and digestibility. Overall, these approaches can be used to optimize crops for
yield and nutritional attributes, which is critical in light of our limited arable land, and the need to feed
a rapidly expanding human population (see also Narrative on Plant Genetics by Ronald).
approaches can be used to optimize crops for yield and nutritional attributes
CRISRP-Cas approaches are also being used to breed next-generation livestock with desirable attri-
butes such as the absence of horns in cows or lowering the fat content in pigs. Recent advances in
42
genetics and breeding for pork, dairy, beef, and poultry open new avenues for increasing resistance
to diseases that afflict livestock, including African swine fever (ASF), bovine respiratory disease (BRD),
and porcine reproductive and respiratory syndrome (PRRS). For example, in the case of PRRS, efforts
are underway to remove a region in a membrane receptor that allows the virus to enter the cell.
Also, this technology can be implemented in industrial bacteria, yeast, and algae used in the bioin-
dustry for the genesis of a wide diversity of products that include commercial biomolecules, drugs,
enzymes, and biofuels.
Although CRISPR-based technologies are already powerful and having significant impacts in the med-
ical, agricultural, and biotechnological industries, it is important to note that this relatively new tech-
nology is still being enhanced and optimized by many scientists around the world. Of note, major goals
include enhancing the efficiency and specificity of the Cas machinery, developing strategies to deliver
the CRISPR-Cas9 machinery to the desired cells and tissues, and improving our ability to predict and
control DNA repair mechanisms and outcomes. There are many innovations still to come.
It is also important to keep in mind that the translation of scientific knowledge into valuable technolo-
gies with commercial applications hinges on regulatory approval and public acceptance. Thus, imple-
mentation of these new technologies must proceed with caution and also consider ethical implications.
It will be critical to engage the public on these issues via an open and transparent dialogue.
Closing Thoughts
I have been privileged to participate in, along with colleagues and collaborators, some of the most
exciting observations and discoveries of the early CRISPR work. I have also enjoyed witnessing
CRISPR technologies being translated into actual products impacting medicine, agriculture, and
biotechnology. I believe that CRISPR will be viewed as one of the most impactful and disruptive
technologies that have emerged in the history of the life sciences.
I am in awe of the speed and scale at which the CRISPR story has unfolded. In merely a decade,
CRISPR has evolved from a study of peculiar regions in bacterial genomes, to an intriguing immune
system in bacteria, and now a potent molecular machine able to readily alter the genomes of humans,
other animals, plants and bacteria at will. CRISPR research is still unfolding at a frenetic pace. The
global impact and rate of adoption of this technology is illustrated by the dissemination of CRISPR
constructs by Addgene, a not-for-profit repository, which has shipped over 100,000 CRISPR-related
plasmids to over 5,000 laboratories in over 100 countries, making this technology broadly available to
all. Consequently, approximately one million scientists are currently developing next-generation gene
therapies for medicine, enhancing desirable attributes of plants and livestock for agriculture, and engi-
neering novel microbes for biotechnology. This is only the beginning of the CRISPR era.
With so much at stake and so many fields impacted by this powerful technology, however, there
is a need for scientists to be mindful of the societal implications of genome editing, especially with
regards to editing humans, and the germline in particular. Several scientific societies have already
43
engaged in an open dialogue to consider the ethical implications of human germline modification and
several countries have implemented a moratorium preventing these applications. Medical and scientific
boundaries—guidelines and guardrails—need to be agreed upon by a diverse group of stakeholders,
and account for concerns about modifying humankind and impacting future generations of individuals.
there is a need for scientists to be mindful of the societal implications of genome

editing
Commercial implementation of this technology in agriculture hinges on regulatory frameworks that
allow the use of CRISPR for genome editing and, thus far, different regulatory agencies have differed
in their approach to regulating editing technologies. Countries like the United States and Japan have
elected not to regulate gene editing for agriculture, whereas the European Court of Justice has ruled
that edited crops fall under the existing genetically modified organism guidelines that restrict commer-
cialization in the European Union. These are reminders that, while scientists rapidly advance on the
technological highway and open new avenues for medicine, agriculture and biotechnology, the actual
development and commercialization of therapies, crops, livestock, and biological products will depend
on a supportive and enabling regulatory network, as well as consumer acceptance. Given the widely
differing public opinions and diverse regulatory agencies operating across the globe, it is still unclear
how easily and quickly these therapies and products will be made available to humankind on a global
basis. With some widespread skepticism about science in general, and unfounded reservations about
genetic engineering, in particular, we are reminded that sometimes consumer opinions can be a difficult
hurdle to overcome for powerful but disruptive technologies.
The CRISPR story that has unfolded over the past decade is also interesting from a scientific
narrative standpoint. The rise of this topic from its discovery in bacteria to scientific stardom in a
relatively short time-span illustrates how rapidly scientific advances can develop, fueled by inter-
disciplinary collaborative teams that transcend the boundaries between industry and academia.
There are also many examples of how serendipity and lucky timing can drive the scientific process
and propel a field forward. Finally, it is important to remember that none of the CRISPR pioneers
could have predicted at the onset that studying a bacterial immune system would lead to new
strategies for treating and potentially eliminating some of the most devastating human diseases.
This is a theme that has emerged over and over again in the history of science—that great new
technological advances start humbly with a curiosity for how life works, and then evolve through
serendipitous observation and putting clues together, step-by-step.
44
Dig Deeper 1:
Strategies for resistance to phage infection: beyond CRISPR immunity.
In the past few decades, advances in Microbiology and Molecular Biology have enabled scientists
to observe and dissect the interplay between phage and bacteria, and characterize the defense
systems that enable microbes to escape viral predation. CRISPR is one among many strategies that
bacteria use to evade viral attacks. Some bacteria fight phage infection by expressing enzymes
that destroy non-self DNA (this is known as a restriction-modification system). In other cases, a
phage-infected bacterium may activate a program that causes it to die prematurely, thereby limiting
the ability of the phage to replicate and infect other bacteria in the population. This kind of self-sac-
rifice to protect the rest of the population is called abortive infection. Mutations in the bacterial
proteins that phage use to attach to the bacterial cell surface can also impede or block phage infec-
tion. The relative contribution of these various phage resistance mechanisms varies widely across
phylogenetic groups of bacteria. In the case of Streptococcus thermophilus, the large majority of the
survivors of phage infection are typically those that have acquired CRISPR-mediated immunity. This
species is particularly enriched in CRISPR-Cas systems: in the thousands of strains tested globally
in the dairy industry, 100% encode CRISPR-Cas systems. Remarkably, these genomes typically
encode multiple CRISPR-Cas systems of different types that can all be concurrently active. Indeed,
there are several reports in the literature showcasing how the CRISPR1 and CRISPR3 CRISPR-Cas
systems in dairy bacteria can concurrently acquire spacers from the same phages.
The CRISPR-Cas story, in which a phage defense system was repurposed to fuel the genome
editing revolution, is reminiscent of a previous chapter of the history of molecular biology. In the
1960s and 1970s, details of the restriction-modification phage defense systems mentioned
above were uncovered, and repurposed as restriction enzymes to drive the recombinant DNA
revolution. As we move forward and continue to mine bacterial genomes for novel CRISPR-Cas
systems, we are reminded that the next “CRISPR” might be awaiting discovery. Indeed, some
recent work has already shown that other novel defense systems exist, which may be the
basis for the development of tools that will fuel the next revolution in biology.
Dig Deeper 2:
Publishing of the Journey to Discovery paper in Science
Altogether, results from these three experiments were filed at the USPTO in the summer of
2006 to convert the aforementioned patent, and it took Philippe and myself 6 months to con-
vince the Danisco management and the reviewers that these results were worth publishing.
At the same time as we were doing our work, other scientists were independently assembling
complementary pieces of the CRISPR puzzle: the paper by Mojica et al., published in 2005,
45
revealed that CRISPR spacers show homology to viruses and plasmids; the paper by Makarova
et al. in 2006 suggested that CRISPR-Cas may be bacterial immune systems. This group of
papers reflects how independent groups can often take similar paths in parallel. Our Danisco
team was ideally positioned to assemble this puzzle given the organism on which we worked,
the presence of an active CRISPR-Cas system, the availability of genomic sequences for both
the host and the viruses, the availability of historically relevant biological material, and per-
fect timing in the sense that some of the clues were already available to put us on the right
trail, while it was obscure enough for others to remain in the darkness.
In addition to the difficulties we internally encountered in our efforts to convince the Danisco
team to spend time and allocate resources to prepare a manuscript for submission to a journal like
Science, we also had to convince the editors and reviewers at the journal that our work was
worthy of their consideration. Initially, the Science reviewers showed relatively little interest
in work on a peculiar genetic locus from a commercial yoghurt starter culture. However, yet
again, luck manifested itself in the shape of timing. Actually, the delay between our initial
patent filing and the original manuscript submission could not have been more fortuitously
productive. Unbeknownst to us, just a couple of weeks before we submitted our paper, the
famous Jill Banfield had submitted a similar story to the very same editor, at the same journal,
discussing circumstantial interplay between CRISPR loci and viral sequences. She observed
this interplay in metagenomic data she had analyzed from acid mine drainage environments
in which bacteria co-evolved with viruses. Luckily for us, she had raised interest at the journal on
this very topic at this very point in time, and I believe this is a primary reason why our paper
was originally reviewed and eventually published.
Dig Deeper 3
Other CRISPR-Cas systems in bacteria
There are many diverse CRISPR-Cas systems, reflecting both the natural diversity and divergent
evolution of the many bacteria that exist and have evolved over time (see Figure DD3). In all
of these systems, immunity is DNA-encoded (invasive DNA is incorporated as spacers into the
CRISPR array), RNA-mediated (involving the production of crRNAs) and nucleic acid-targeting
(the systems specifically recognize nucleic acid sequences complementary to the crRNA).
In general, CRISPR-Cas systems are split into two main classes, six major types, and 33
sub-types that vary in their biochemical processes. For instance, class 1 systems rely on a
multi-protein complex to carry out the interference process, whereas class 2 systems use a
single protein to target and cleave nucleic acids. Within each class, there are different types
that are each defined by a signature Cas protein, which is responsible for a specific type of
nucleic acid cleavage as follows:
46
•T ype I: the Cas3 exonuclease in complex with Cascade (CRISPR associated complex for
antiviral defense) targets double-stranded DNA (dsDNA) using PAM targeting on the 5¢
edge of the protospacer. The exonuclease selectively nicks and chews the target strand
in a 3¢ to 5¢ exo-nucleolytic manner, creating extensive damage in the viral DNA. This is
likely why this “Pacman-type” system is the most widespread in nature.
• Type II: the Cas9 endonuclease targets dsDNA and the RuvC and HNH nickase domains
each nick one DNA strand and generate a blunt cut. This involved PAM targeting at the
3¢ end of the protospacer. This system generates a clean DNA cut (a double-stranded
break—DSB), which is why Cas9 is often referred to as a molecular scalpel and is useful
for triggering DNA repair pathways linked to genome editing.
• Type III: the Cas10 nuclease targets single-stranded RNA (ssRNA) and together with the
Csm or Cmr complex cleaves the target RNA at multiple locations. There is no depen-
dence on a PAM sequence in this type.
• Type V: the Cas12 endonuclease nicks two strands of dsDNA at different locations to
generate sticky ends with overhangs. This involves a PAM flanking the 5¢ edge of the
protospacer.
• Type VI: the Cas13 nuclease nicks complementary ssRNA and also carries a non-specific
RNase activity that creates “collateral damage.”
Figure DD3: Different types of Cas protein found in the bacterial kingdom that destroy DNA,
RNA (or both) in different ways.
47
Dig Deeper 4:
Correcting the genetic defect of DMD
The Dystrophin gene is extraordinarily large, comprised of >3,500 amino acids. Dystrophin
is divided into 79 exons with a corresponding number of intervening introns. DMD mutations
disrupt the dystrophin reading frame, introducing premature stop codons that generate truncated
and unstable protein. (See Dig Deeper 4 Video for a description of exon, introns and RNA
splicing). There are thousands of different types of DMD mutations, but many DMD mutations
ultimately cause a shift in the reading frame starting from exon 51. Therapies that restore
the reading frame at, or after, exon 51 could benefit ~13% of DMD patients. The DMD dog is
a model for just such a mutation: exon 50 is missing, and the resulting fusion of exon 49 to
exon 51 creates a frameshift that generates a stop codon in exon 51 (see the figure).
Dig Deeper 4 Video. RNA splicing
Viral vectors were designed to target the CRISPR/Cas9 machinery to exon 51, and the result-
ing viruses were injected into the muscle of DMD dogs. The Cas9 molecular scalpel was
guided to the exon 51 sequence, where it generated a double-strand break that recruited
the DNA repair machinery. NHEJ repaired the break, which generated a variety of insertions,
deletions, and substitutions. Many of these did not fix the frame shift, but since the gene is
already non-functional, they did not do any additional harm. However, a relatively frequent
consequence of this repair was that a single nucleotide was added at the cut site. This one
nucleotide insertion restored the wild-type reading frame, so that the gene could now produce
functional protein to support muscle function.
48
References and Resources

Guided Papers
Barrangou, R., et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science
2007;315:1709–1712.
Amoasii, L. (). Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.
Science 362: 86-91.
Other References
Ishino, Y. Shinagawa, H, Makino, K., Amemura, M. and Nakata, A. Nucleotide sequence of the iap gene,
responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene
product. J. Bacteriol. 1987;169:5429–5433.
The first description of the CRISPR sequence described in Clue 1 of the Journey to Discovery.
Mojica, F.J., Diez-Villasenor, C, Garcia-Martinez, J., and Soria, E. Intervening sequences of regularly spaced
prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 2005;60:174–182.
This paper shows a connection between spacer sequences in CRISPR and viral sequences, as discussed
in Clue 2 of the Journey to Discovery.
Markarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I., and Koonin, E.V. A putative RNA-interference-based
immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies
with eukaryotic RNAi, and hypothetical mechanism of action. Biol. Direct. 2006;1:7.
This paper makes a connection of the Cas genes with a possible defense mechanism for bacteria, as
discussed in Clue 2 of the Journey to Discovery.
Other Resources
1. CRSPR-Cas9 Video. Available at: https://www.youtube.com/watch?v=2pp17E4E-O8 A short video that
gives a basic explanation of how the CRISPR-Cas9 system functions in bacteria and how scientists
have adapted the system to work in other organisms.
2. Q and A’s with Rodolphe Barrangou. Available at: https://www.pnas.org/content/114/28/7183

A CRISPR centric question and answer session with Dr. Barrangou done by PNAS.
3. Dr. Barrangou Lecture. Available at: https://vimeo.com/255061822

A video of Dr. Barrangou giving a lecture on how CRISPR was discovered, the research he has done and
the implications of the technique for the future in the context of plant research.
4. CRISPR Short Video. Available at: https://www.youtube.com/watch?v=9IgLrOEsauk

A short video featuring Dr. Barrangou explaining the CRISPR system and it’s implications.
5. CRISPR FAQ. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-

and-answers-about-crispr. A web page that includes a short video explaining the CRISPR-Cas9 system
and has a series of frequently asked questions and their answers.
49
6. CRISPR-Cas9 Mechanisms and Applications. Available at: https://www.hhmi.org/biointeractive/crispr-

cas-9-mechanism-applications. An interactive simulation of both how the CRISPR-Cas9 system functions
and what the real world applications are.
7. CRISPR Timeline. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-

spotlight/crispr-timeline
A webpage that displays a timeline of the major developments in CRISPR research as well as brief
descriptions of the work that was done and the impact of that work.
8. CRISPR Slide Show (PDF). Available at: http://ilsi.org/wp-content/uploads/2016/07/7.-BARRANGOU-

Rodolpe-CRISPR-Cas.pdf
This is a PDF presentation slides created by Dr. Barrangou discussing CRISPR and it’s impacts on the
world of food science research.
9. Jennifer Doudna CRISPR Explanation. Available at: https://www.youtube.com/watch?v=SuAxDVBt7kQ

A video featuring Dr. Jennifer Doudna explaining how they discovered the CRISPR-Cas9 system, how it
works and the directions it is moving today.
10. Jennifer Doudna CRISPR TED Talk. Available at: https://www.youtube.com/watch?v=TdBAHexVYzc

A TED talk video that features Dr. Jennifer Doudna explaining the discovery, potential functions and eth-
ical issues surrounding the CRISPR-Cas9 system.
11. Lecture on DNA repair by Jim Haber (Brandeis University) on iBiology.org. Available at: https://www.
ibiology.org/genetics-and-gene-regulation/mechanisms-dna-repair/#part-1
50

CRISPR Cas Bacterial Adaptive Immunity

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CRISPR Cas:

From Bacterial Adaptive Immunity to a

A Narrative produced by The Explorer’s Guide to Biology

Version Date: September, 2019

Terms and Concepts Used

Terms and Concepts Explained

Part I: Journey to Discovery—CRISPR-Cas systems provide adaptive immunity

Part II: Knowledge overview—How widespread adaptive immunity in bacteria and

Part I: The Journey to Discovery

Figure 2. The role of bacterial fermentation in making yoghurt.

Clue 1: CRISPR sequences in bacterial genomes

Clue 2: CRISPR sequences are paired with cas genes

Video 1. Whiteboard on Bioinformatics

Clue 3: CRISPR “Spacer” sequences show homology to phage DNA

The Discovery: CRISPR-Cas is a bacterial immune system

Step 1: Clues from Analyzing DNA Sequences of the CRISPR Locus

Explorer’s Question: What might this result suggest to you?

The CRISPR Locus has Homology to Viral Sequences

Figure 6. A nucleotide

Step 2: Experiments to Test Whether CRISPR Serves an Adaptive Immune System

CRISPR loci acquire phage sequences

In our experiment, we selected phage-resistant variants (colonies of bacteria that survived on

CRISPR spacers confer phage resistance

A) adding a spacer: phage resistance was acquired;

Cas genes are involved in acquisition and interference

What happened next?

With CRISPR-Cas, scientists now have a tool to “rewrite” the genome

Starting from humble beginnings – curiosity about patterns of nucleotides in the

Part II: Knowledge Overview

Video 3. A summary of how CRISP immunity works by Rodolphe Barrangou.

How CRISPR immunity works

Repurposing Cas molecular machines for genome editing

Figure 15. The interfer-

The trick is how to cut the DNA at a defined location

In practice, genome editing works as follows (see Figure 18):

Other Uses of CRISPR-Cas9

Part III: Frontier

The method is efficient, relatively easy and relatively inexpensive, democratizing

CRISPR-Cas9 and the treatment of Duchenne Muscular Dystrophy

Figure 19. Examples of CRISPR-Cas9 genome editing applications.

Video 5. Whiteboard video on RNA Splicing.

To study human disease, scientists often create a genetic defect in an animal

Other examples of uses of CRISPR-Cas technology

there is a need for scientists to be mindful of the societal implications of genome

Dig Deeper 4 Video. RNA splicing

References and Resources

2. Q and A’s with Rodolphe Barrangou. Available at: https://www.pnas.org/content/114/28/7183

3. Dr. Barrangou Lecture. Available at: https://vimeo.com/255061822

4. CRISPR Short Video. Available at: https://www.youtube.com/watch?v=9IgLrOEsauk

5. CRISPR FAQ. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-

6. CRISPR-Cas9 Mechanisms and Applications. Available at: https://www.hhmi.org/biointeractive/crispr-

7. CRISPR Timeline. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-

8. CRISPR Slide Show (PDF). Available at: http://ilsi.org/wp-content/uploads/2016/07/7.-BARRANGOU-

9. Jennifer Doudna CRISPR Explanation. Available at: https://www.youtube.com/watch?v=SuAxDVBt7kQ

10. Jennifer Doudna CRISPR TED Talk. Available at: https://www.youtube.com/watch?v=TdBAHexVYzc

You might also like

2. Q and A’s with Rodolphe Barrangou. Available at: https://www.pnas.org/content/114/28/7183

3. Dr. Barrangou Lecture. Available at: https://vimeo.com/255061822

4. CRISPR Short Video. Available at: https://www.youtube.com/watch?v=9IgLrOEsauk

5. CRISPR FAQ. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-

6. CRISPR-Cas9 Mechanisms and Applications. Available at: https://www.hhmi.org/biointeractive/crispr-

7. CRISPR Timeline. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-

8. CRISPR Slide Show (PDF). Available at: http://ilsi.org/wp-content/uploads/2016/07/7.-BARRANGOU-

9. Jennifer Doudna CRISPR Explanation. Available at: https://www.youtube.com/watch?v=SuAxDVBt7kQ

10. Jennifer Doudna CRISPR TED Talk. Available at: https://www.youtube.com/watch?v=TdBAHexVYzc