Genome editing and crispr cas 9

One of the most exciting recent developments in genetic engineering is CRISPR-Cas9

(CRISPR). CRISPR derives its name from "clustered regularly interspaced short palindromic
repeats," genomic sequences that microbes use to defend themselves against viral attacks.
Along with CRISPR associated (Cas) proteins, bacteria use the sequences to recognize and
disarm future invading viruses. Scientists have adopted this system for use in genetic
engineering.
CRISPR-Cas technology allows scientists to edit genes and manipulate gene expression with
a level of ease that was not possible using other methods. Importantly, it also allows
researchers to edit genes within living organisms, a fact that supports the use of CRISPR-Cas
in a far-reaching range of applications from basic research to the development of novel
therapies and other biotechnology products.
The 2020 Nobel Prize for Chemistry was awarded to Emmanuelle Charpentier and Jennifer
Doudna for the development of this method for genome editing.
This page contains background information about the CRISPR technology as well as other
resources and activities. Please also watch our video, CRISPR Explained: Gene Editing History,
Technology, and Applications.

CRISPR-Cas — A Microbial Immune System

Humans have complex immune systems that involve the coordinated activities of multiple
cell types, organs, and signaling systems to recognize and respond to active infections.
Prokaryotes — bacteria and archaea — also have a form of adaptive immunity that allows
them to recognize and respond to viral infections.

Bacterial Chromosome A CRISPR region within a microbial genome.

Some prokaryotic genomes contain short, palindromic DNA sequences that are repeated many
times, with unique "spacer" sequences between the repeats. These "clustered regularly
interspaced short palindromic repeats" (CRISPR) are followed by short segments of spacer
DNA that match DNA sequences found in bacteriophage genomes (bacteriophage are viruses
known to infect bacteria). Groupings of CRISPR-associated (Cas) genes are also found next to
CRISPR sequences, and these Cas genes encode enzymes that cut DNA in specific places,
like precise molecular scissors.
CRISPR-Cas9 sequences work together to provide an immune response in which a microbe
could recognize invading DNA and unleash Cas enzymes to cut and disarm it. CRISPR
sequences and the Cas enzymes are the keys to the microbial "adaptive immune response,"
which involves three phases:
● Cutting and Capture — When bacteria are infected by a virus, they use their CRISPR
system to cut up the invading viral DNA and insert pieces of it (spacers) into their own
genome as a "memory" of the infection
● Monitoring — Bacteria transcribe the spacers into RNA, which can form a complex
with the Cas9 enzyme. These complexes monitor the cell for any DNA sequence
complementary to the RNA
● Defense — If matching (viral) DNA is encountered, the spacer RNA-Cas9 complex
binds to it and cuts the viral DNA to prevent it from replicating. This halts the viral
infection

Using this system, bacteria can collect sequences from many different infecting viruses to create
a "library." Since the CRISPR sequence is contained in genomic DNA, it is passed on to each
generation, and the library continues to change and adapt to more common threats over time.
●
Acquire Foreign DNA Sequences
 ●
Defend Against Future Infection

The CRISPR-Cas9 microbial defense system. 1. The Cas1-Cas2 enzymes of the microbe recognize
and cut out a segment of foreign DNA. 2. The Cas1-Cas2 enzymes insert the DNA segment into the
CRISPR region of the bacterial genome as a spacer. 3. A spacer sequence is transcribed and then
linked to a Cas9 protein. 4. Upon reinfection by the same invader, the CRISPR-Cas9 complex can
recognize the foreign DNA sequence and cut it to prevent complete reinfection.

CRISPR-Cas9 — A Gene
Editing System
The CRISPR-Cas9 system uses modified components of the bacterial CRISPR system to
direct target-specific cutting of double-stranded DNA. DNA repair mechanisms then take over
to fix the break in a manner that modifies the genetic sequence that has been cut.

Cutting the DNA

● Cas9 enzyme (Cas9) — an endonuclease that cuts both strands of DNA at a specific
site. Multiple types of Cas enzymes are found in nature, but Cas9 is commonly used in
the laboratory
● Single guide RNA (sgRNA) — an engineered RNA that forms a complex with Cas9.
The sgRNA is a fusion of two regions that occur as separate RNAs in nature:
● Guiding region — part of the CRISPR RNA or crRNA in nature, a 20-nucleotide
region that is complementary to the target region and defines the target DNA
sequence that Cas9 cuts. Scientists customize this sequence for their own
targets
● Scaffold region — the trans-activating CRISPR RNA or tracrRNA in nature, this
region forms a multi-hairpin loop structure (scaffold) that binds in a crevice of
the Cas9 protein

● Protospacer adjacent motif (PAM) — required for Cas9 function, this sequence motif
is immediately downstream of the target sequence. Cas9 recognizes the PAM
sequence 5’-NGG, where N can be any nucleotide (A, T, C, or G). When Cas9 binds the
PAM, it separates the DNA strands of the adjacent sequence to allow binding of the
sgRNA. If the sgRNA is complementary to that sequence, Cas9 cuts the DNA

5 Steps of Cas9 DNA Cleavage

●

● 5. The complex releases from the DNA

● The Cas9-sgRNA complex releases the cut DNA and is ready to repeat the process.

● 1. Cas9 Binds an sgRNA

● Cas9 recognizes and binds the scaffold (tracrRNA) region of a sgRNA. The nucleotide
sequence of the scaffold region determines its structure, which is tailored to fit within
the Cas9 protein as a key fits into a lock.
●

● 2. The Cas9-sgRNA complex binds to a PAM site on

the target DNA
● Cas9 requires a particular PAM sequence (5’-NGG) to be present directly adjacent to
the protospacer sequence. When the Cas9-sgRNA complex recognizes and binds a
PAM, it separates the DNA strands of the adjacent sequence to allow binding of the
sgRNA.

● 3. The guiding region of the sgRNA binds to the

target DNA sequence
● The guiding region of the sgRNA attempts to base-pair with the DNA. If a match is
found, the process continues. Otherwise, the complex releases and attempts to bind
another PAM and target DNA sequence.

● 4. Cas9 makes a double-stranded break in the DNA

three base pairs upstream of the PAM

● 5. The complex releases from the DNA

● The Cas9-sgRNA complex releases the cut DNA and is ready to repeat the process.
●

● 1. Cas9 Binds an sgRNA

● Cas9 recognizes and binds the scaffold (tracrRNA) region of a sgRNA. The nucleotide
sequence of the scaffold region determines its structure, which is tailored to fit within
the Cas9 protein as a key fits into a lock.
Repairing the Break to Engineer the Change
Researchers can use the cell’s own DNA repair machinery to modify, insert, or delete a
nucleotide sequence. The repair can happen in two ways:

● Non-homologous end joining (NHEJ) — enzymes reconnect the ends of the

double-stranded break back together. This process may randomly insert or delete one
or more bases and can cause mutations that can disrupt gene function or expression
● Homology directed repair (HDR) — proteins patch the break using donor template
DNA. Researchers design the donor template DNA that may include a desired
sequence flanked on both sides by "homology arms" that match the sequence
upstream and downstream of the cut. A complementary DNA strand is created during
the repair
DNA repair via homology directed repair and non-homologous end joining

Applications of CRISPR
Technology
With CRISPR, targeted disruption of any gene — in most organisms — is possible. It allows
scientists to modify genomic DNA with precision to ensure that no other genes or sequences
are unintentionally disrupted. CRISPR technology is easier, faster, and less expensive than
other gene-editing techniques and can be used to edit multiple genes at the same time in a
single cell. Finally, CRISPR requires the introduction of only one protein (Cas9) and one sgRNA
into a cell. Such a powerful technology can be expected to have a vast range of applications.

Medicine
Researchers are looking to CRISPR as a technique for editing out genetic defects that result in
sickle cell disease, cystic fibrosis, hemophilia, and muscular dystrophy, and for developing
more targeted and effective cancer treatments. One study showed that adult rats engineered
to have a genetic form of blindness could be treated using CRISPR gene therapy (Berry et al.
2019). The goal is to someday have patients' diseased cells removed, "fixed" with CRISPR, and
then returned to their bodies to treat various conditions or have diseased organs be treated
directly with CRISPR. The potential for using CRISPR to change genetic traits in humans has
raised serious concerns, about possible unintended effects, as well as ethical questions. The
ease of applying CRISPR has caused worry about the potential misuse of the technology.
Despite these concerns, CRISPR is revolutionizing many aspects of biotechnology and
scientific research.

Agriculture
CRISPR technology is expected to accelerate the development of new, improved crops. The
technology has produced crops and livestock with desirable traits such as faster growth,
higher nutrient content, and disease resistance. And, since CRISPR technology can modify
genes without introducing new genes, CRISPR-modified plants may be subjected to lighter
regulations than other genetically modified crops.

Industry
Scientists have used a modified CRISPR-Cas9 system to create a yeast strain to produce
lipids and polymers. These molecules could be useful in the development of biofuels,
adhesives, and fragrances. Currently, these lipids and polymers are made synthetically from
non-renewable petroleum-based materials that are more expensive and could present safety
risks.

Public Health
Scientists are experimenting with using CRISPR to engineer "gene drives" to spread specific
genes through a population of insect pests that cause them to die or become infertile. This
technique is being considered to eradicate mosquitoes carrying human pathogens like
malaria parasites or Zika virus.