Unit 4.

Gene editing. CRISPR.

SECTION II: CELL STRUCTURE AND FUNCTION.
GENE EDITING

Previous
technology:

Recombinant DNA
Genetic editing: Recombinant
Gene editing
DNA

Modifications directed
at the genome, their
contexts (epigenetic
marks) or their results
(transcripts), using
endonucleases *

*Endonuclease: enzyme that cuts DNA

Gene editing tools

➢ ZFNs (Zinc Finger Nucleases)

High cost and difficulty, operating problems
➢ TALENs (Transcription Activator-Like
Effector Nucleases)
More difficult and expensive than CRISPR
➢ CRISPR-Cas
What is CRISPR-Cas?

Natural defense system against

viruses in bacteria and archaea
• CRISPR: Clustered Regularly
Interspaced Short Palindromic
Repeats
• Cas: CRISPR-associated
The natural system acts in 3 phases:
Acquisition, expression and interference
CRISPR as a gene
editing tool
Francisco Martínez Mojica

1993. Describes repeated DNA sequences in

archaea Haloferax mediterranei

2000. Same sequences in 20 different

microorganisms

2005. The biological function of CRISPR is

intuited
 2012. Emmanuelle Charpentier
and Jennifer Doudna elucidated
the mechanism of CRISPR/Cas 9
and demonstrated that it had
potential use for gene editing
2013. Feng Zhang uses
CRISPR-Cas9 on the genome
of a living mammalian cell
 8000

7000

6000

5000
Number of publications

4000

3000

2000

1000

0
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
Year

Historical distribution of CRISPR documents available in Pubmed

How does the technique work?
DNA repair mechanisms

▪ nonhomologous end-joining
▪ homology-directed repair
APPLICATIONS
 Medical applications
❑GERMINAL GENETIC MODIFICATION
• In reproductive cells (sperm and eggs), which are modified
by introducing functional genes into their genomes or by
disrupting the wrong genes
• In early embryonic development

Germ gene editing leads

to permanent changes
that are passed on to the
next generations
1. Liang P, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. 2015.
2. Kang X, et al. Introducing precise genetic modifications into human 3PN embryos by
CRISPR/Cas-mediated genome editing. 2016.
3. Tang L, et al. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. 2017.
4. Ma H, et al. Correction of a pathogenic gene mutation in human embryos. 2017.
5. Fogarty NME, et al. Genome editing reveals a role for OCT4 in human embryogenesis. 2017.
6. Li G, et al. Highly efficient and precise base editing in discarded human tripronuclear embryos.
2017.
7. Zhou C, et al. Highly efficient base editing in human tripronuclear zygotes. 2017.
8. Liang P, et al. Correction of β-thalassemia mutant by base editor in human embryos. 2017.
9. Tang L, et al. Highly efficient ssODN-mediated homology-directed repair of DSBs generated by
CRIPSR/Cas9 in human 3PN zygotes. 2018.
10.Zeng Y, et al. Correction of the Marfan syndrome pathogenic FBN1 mutation by base editing in
human cells and heterozygous embryos. 2018.
11.Zhang M, et al. Human cleaving embryos enable robust homozygotic nucleotide substitutions
by base editors. 2019.
12. Li et al. Efficient generation of pathogenic A-to-G mutations in human tripronuclear embryos via
ABE-mediated base editing. 2019.
The first experiment to modify the genome of human embryos is authorized in Spain

https://www.observatoriobioetica.org/2020/02/se-autoriza-el-primer-experimento-
de-modificacion-del-genoma-de-embriones-humanos-en-espana/32621
November 2018
• Two twin girls were born,
Lulu and Nana, whose DNA
was modified with
CRISPR/Cas to give them
'protection' against HIV
• The research has not been
published in any scientific
journal, so its data could not
be verified
• "It's irresponsible," say
scientists
He Jiankui photographed in his lab
Germinal genetic editing. Ethical aspects
➢ Are the risks acceptable?
➢ Is the destruction of thousands of human embryos acceptable?
➢ What happens with informed consent?
➢ Therapy or reproductive option?
➢ Can it lead to a less inclusive society and situations of
discrimination?
➢ Is it ethical to produce “designer” children?
➢ Association with IVF
Are the risks acceptable?
• Off-target effects

• Repair by NHEJ route: random insertions or deletions

(mutations on target, low efficiency HDR route)

• Mosaicism
 “the gene-edited embryos were mosaic. For
example, embryo No. 16 contained many different
kinds of alleles”
(Liang P, et al. CRISPR/Cas9-mediated gene editing in human
tripronuclear zygotes. 2015)

“Because the edited embryos are genetically mosaic,

it would be impossible to predict gene editing
outcomes through pre-implantation genetic
diagnosis (PGD)”
“Taken together, our work highlights the
pressing need to further improve the
fidelity and specificity of the
CRISPR/Cas9 platform, a prerequisite
for any clinical applications of
CRSIPR/Cas9-mediated editing” (1)
 “we did not detect off-site targeting as
predicted for a total of 28 potential off-target
sites. Recent studies used various strategies
to experimentally map off-target mutations of
the CRISPR/Cas system and found that
computational prediction based on
sequence similarity to target sites failed to
predict many off-target mutations.
Therefore, the embryos likely carried off-target
mutations undetected by our genotyping” (2)
“the use of CRISPR/Cas9-mediated
gene editing in reproductive clinics is
not a current option due to both
ethical and technical issues” (3)
“Although we did not find off-target
effects at the top 10 potential off-target
sites examined, the specificity of
base editors needs more
comprehensive investigation
through genome-wide specificity
assays” (8)
1. Alanis-Lobato, G. et al. Preprint at bioRxiv
https://doi.org/0.1101/2020.06.05.135913 (2020).
2. Zuccaro, M. V. et al. Preprint at bioRxiv
https://doi.org/10.1101/2020.06.17.149237 (2020).
3. Liang, D. et al. Preprint at bioRxiv
https://doi.org/10.1101/2020.06.19.162214 (2020).
Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos.
Alanis-Lobato et al. bioRxiv 2020.06.05.135913; doi:
https://doi.org/10.1101/2020.06.05.135913

Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.

Zuccaro et al. Cell. 2020 Dec 10;183(6):1650-1664.e15. doi: 10.1016/j.cell.2020.10.025.
Epub 2020 Oct 29. PMID: 33125898.

Frequent gene conversion in human embryos induced by double strand breaks.

Liang et al. bioRxiv 2020.06.19.162214; doi: https://doi.org/10.1101/2020.06.19.162214
"If human embryo editing for
reproductive purposes or germline
editing were space flight, the new
data are the equivalent of having
the rocket explode at the launch pad
before take-off"

Fyodor Urnov, estudia edición del genoma en la

Universidad de California, Berkeley,

https://www.nature.com/articles/d41586-
020-01906-4
Human embryos destruction
Ref. 3PN embryos 2PN embryos
1 86 0
2 213 0
3 99 20
At least 866
4 0 167
human
5 0 54
embryos
6 25 0
7 68 0
destroyed in
8 0 65
10 papers
9 23 0
10 0 46
11 ? ?
TOTAL = 514 352
➢About informed consent: inability of the embryo to give it, will affect
all its offspring, will be the subject of research
➢Therapy or reproductive option? The treatment is foreseen before
the conception of the "patient". Questions about allocation of public
resources.
➢Can it lead to a less inclusive society and situations of
discrimination? Private exploitation of these techniques can lead to
higher and lower genetic levels. Also, "genetic imperfection" would
be a choice that would be borne by the "irresponsible" parents.
➢Is it ethical to produce “designer” children? Germline gene
editing opens the door to human genetic improvement
➢Association with IVF
Resource
allocation
In which cases is there no alternative for parents?

4-8 x 10-8 % Couples for a certain gene.

Cases of severe monogenic diseases in which all children would
inherit the genotype of the disease.

Autosomal Autosomal X-linked recessive

dominant disease recessive disease diseases
If one parent carries two If both parents carry two If the expectant mother
disease-causing alleles disease-causing alleles carries two disease-
(homozygous affected), in the same gene causing alleles
all children will inherit (homozygous affected), (homozygous affected)
the disease-causing all children will inherit and the male father
genotype. the disease-causing carries one disease-
genotype. causing allele on his
only X chromosome
(hemizygous affected),
all the offspring would
be affected.
Jiankui case specifications
• Falsified ethical approval documents
• Totally unfavorable benefit risk balance.
• He chose a target disease that can be prevented and
treated.
• Chose a genetic target, the CCR5 gene, with important
functions (risks)
• His experiment cannot be considered therapeutic, it is
an "improvement"
• Embryonic destruction
What is the current position of the scientific community?
• National Academies of Sciences, Engineering and Medicine.<<Human
Genome Editing: Science, Ethics, and Governance>>. Washington, DC: The
National Academies Press. 2017.
It is declared that in the future the clinical application of germline
genetic editing could become a realistic option

• Ormond, KE et al. <<Human Germline Genome Editing>>. The American

Journal of Human Genetics. 2017; 101(2): 167-176.
The door is not closed to a possible future use of germline gene
therapy, but it is pointed out that today the unresolved issues make it
inappropriate in embryos for later implantation

• Chan, S et al. <<Genome editing technologies and human germline genetic

modification: The Hinxton Group consensus statement>>. The American
Journal of Bioethics. 2015; 15(12): 42–47.
It requested the continuation of research on embryos but the failure
to carry out any reproductive application
 The same trend can be seen in Europe
• Chneiweiss, H et al. <<Fostering responsible research with genome editing
technologies: a European perspective>>. Transgenic Research. 2017; 26(5): 709–713
In March 2016, representatives from more than 20 European
countries came together to reflect on and promote responsible research with
CRISPR-Cas. In July 2017, the consensus document was published which
also reflected this vision

• de Wert, G et al. <<Human germline gene editing: Recommendations of ESHG and

ESHRE>>. European Journal of Human Genetics. 2018; 26(4): 445-449

• Nuffield Council on Bioethics. <<Genome editing and human reproduction: social and
ethical issues>>. 2018, 136

• Howard, HC et al. <<One small edit for humans, one giant edit for humankind? Points
and questions to consider for a responsible way forward for gene editing in humans>>.
European Journal of Human Genetics. 2018; 26(1):1-11
Recommends conducting careful scientific research to build an
evidence base
 The second international summit on
human genome editing (27-29
November 2018) recommends
charting the path towards germline
gene editing:

“the scientific understanding and technical requirements for clinical practice

remain too uncertain and the risks too great to permit clinical trials of germline
editing at this time. Progress over the last three years and the discussions at the
current summit, however, suggest that it is time to define a rigorous, responsible
translational pathway toward such trials”

Statement by the Organizing Committee of the Second International

Summit on Human Genome Editing
November 28, 2018
https://www.nationalacademies.org/news/2018/11/statement-by-the-organizing-committee-of-the-second-
international-summit-on-human-genome-editing
“more than 30 countries including China already have regulations
and laws in place prohibiting genetic modifications to the human
germline […] a pressing issue is not an additional moratorium or
bans, but how to reinforce already existing regulations around
the world”
(Wolf, Don P. Mitalipov, Paul A. Mitalipov, Shoukhrat M. 2019/06/01. Principles
of and strategies for germline gene therapy. Nature Medicine)
 National Academy of Medicine, National
Academy of Sciences, and the Royal Society.

“Human embryos whose genomes have been

edited should not be used to create a pregnancy
until it is established that precise genomic
changes can be made reliably and without
introducing undesired changes - criteria that have
not yet been met"

National Academy of Medicine, National Academy of

Sciences, and the Royal Society. 2020. Heritable Human
Genome Editing. Washington, DC: The National
Academies Press. https://doi.org/10.17226/25665.
“It is about preventing research that produces serious unwanted effects and,
with it, a social alarm that blocks research in an extraordinarily promising field for
a long time. Neither scientists, nor States, nor international organizations intend to
build firewalls that prevent the creation of genetically modified human beings in the
future. Rather, they have established controls so that, as far as possible, the
territory of the investigation is transited by responsible groups, in the sense that they
do not jeopardize the public's confidence in those investigations. They do not
think about the protection of the human embryo, nor about the risks of ending
up legitimizing gene editing for clinical purposes as well as for improvement
purposes: the only thing that seems to worry is that there is a scandal that will
destroy an area of technological and commercial development with a great future”.

La revolución de la edición genética mediante CRISPR-Cas9 y los desafíos éticos y

regulatorios que comporta. Bellver Capella. 2016. Cuadernos de bioética, 27(90): 223-239
Baylis F, Darnovsky M, Hasson
K, Krahn TM. Human Germ Line
and Heritable Genome Editing:
The Global Policy Landscape.
CRISPR J. 2020 Oct;3(5):365-
377. doi:
10.1089/crispr.2020.0082.
Erratum in: CRISPR J. 2021
Apr;4(2):301-302. PMID:
33095042.
❑SOMATIC GENETIC MODIFICATION
Clinical trials in this
field are few but have In 2015, Layla Richards, a one-year-old girl who at 14 weeks of age
already started, had been diagnosed with aggressive leukemia, received an
especially ex vivo experimental treatment based on TALENs: the so-called UCART19
cells.
trials
CAR-T cell therapy (Chimeric Antigen Receptor T-Cell or chimeric
antigen receptor of T cells) consists of extracting T lymphocytes
from the patient (cells of the immune system). These are later
modified by gene editing to recognize and attack tumor cells, and
are transferred back to the patient's body so that, after being
reprogrammed, they can recognize, attack and destroy cancer cells.

A single 1 ml dose of these cells was administered and within 2

months the cancer cells disappeared. She is currently cancer-free
and has no problems associated with the technique.

More patients are receiving this treatment:

https://clinicaltrials.gov/ct2/show/NCT02808442?intr=UCART19&rank=1

Brian Madeux, first person to receive
in vivo gene editing
Hunter syndrome: due to the deficiency of the
enzyme iduronate-2-sulfatase, there is an
inability of the body to break down and recycle
some sugars. Its accumulation interferes with the
function of certain cells and organs, leading to
the appearance of several serious symptoms
ZFN have been used to correct the error in the
genome causing this disease in at least 4
patients. Early findings suggest that the
technique is safe, but are inconclusive as to
efficacy.
(EDIT-101) is an experimental medicine
delivered via sub-retinal injection under
development for the treatment of Leber
congenital amaurosis 10 (LCA10), an
inherited form of blindness caused by
mutations in the CEP290 gene.
The trial will assess the safety,
tolerability, and efficacy of EDIT-101
in approximately 18 patients with this
disorder.
https://www.nature.com/articles/d41586-020-00339-3
https://science.sciencemag.org/content/367/6481/eaba7365
https://pubmed.ncbi.nlm.nih.gov/32775490/
Somatic genetic editing
Ethical aspects
➢ Biosafety issues

Off-target effects
Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large
deletions and complex rearrangements. Nat Biotechnol. 2018 Sep;36(8):765-771

Immune response to Cas9

Charlesworth, et al. Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans. Posted
in bioRxiv on 5 January 2018

Relationship with cancer

Haapaniemi E, et al. Nat Med. 2018 Jul;24(7):927-930.CRISPR-Cas9 genome editing induces a p53-
mediated DNA damage response

➢ Controlled clinical trials

➢ Proper disease selection

❑PERSONALIZED MEDICINE

Rare diseases, such as albinism, cancer…

Biotechnological
applications

“We should be aware that, if CRISPR technology is going to

influence our lives in any way, it will probably do so sooner
through what we are going to eat and not through new
innovative treatments, which will still be late in coming and
will not do so until the required levels of safety and efficacy
are achieved”.
Lluís Montoliu
❑LIVESTOCK

✓ The piglets subjected to editing lived with

seven other normal conspecifics in the same
pen, and then all of them were inoculated
with the PRRS (porcine reproductive and
respiratory syndrome) virus.

✓ Five days later the ordinary pigs contracted

a fever and fell ill, but the genetically
modified pigs remained healthy

Nat Biotechnol. 2016 Jan;34(1):20-2. Gene-edited pigs are protected from porcine reproductive and respiratory
syndrome virus. Whitworth KM, et al.
CRISPR/Cas9-
mediated generation
of animal models and
application in human
health
❑AGRICULTURE
✓ CRISPR permite
obtener cultivos de
arroz resistentes a
la contaminación
radiactiva,
inactivando el
transporte de cesio
radiactivo
depositado en el
suelo

Plant J. 2017 Oct;92(1):43-56.

Production of low-Cs+ rice plants
by inactivation of the K+
transporter OsHAK1 with the
CRISPR-Cas system. Nieves-
Cordones M, et al.
 Number of genes modified using
CRISPR/Cas system with the aim
of crops improvement

Anna KorotkovaS. V. GerasimovaS. V.

Current achievements in modifying crop genes using
CRISPR/Cas system
February 2019Vavilov Journal of Genetics and Breeding
23(1):29-37
DOI: 10.18699/VJ19.458
CRISPR-edited plants
Soybean (Glycine max) with drought and salt tolerance; achieved by
disrupting the Drb2a and Drb2b genes (double-stranded RNA-binding
protein2 genes)
Camelina with increased oil content; target genes not disclosed
Setaria viridis, or green bristlegrass, with delayed flowering time;
achieved by deactivating the S. viridis homolog of the Zea mays ID1
gene
Waxy corn with starch composed exclusively of amylopectin; achieved
by inactivating the endogenous waxy gene Wx1 that encodes a
granule-bound start synthase catalyzing production of amylose

White button mushroom (Agaricus bisporus) with antibrowning

properties; achieved by knocking out a gene coding for polyphenol
oxidase (PPO)
Nat Biotechnol. 2018 Jan 10;36(1):6-7. With a free pass, CRISPR-edited plants reach
market in record time. Waltz E.
Crops that are made resistant to diseases via CRISPR/Cas9

Crop Disease/symptoms Targeted gene

Triticum aestivum Powdery mildew disease TaMLO-A1 (wheat mildew resistance locus1)
Oryza sativa Bacterial blight of rice
Rice blast disease
Arabidopsis Turnip mosaic virus
thaliana disease
Gossypium Cotton leaf curl disease
hirsutum
Cucumis sativus L Ring spot disease, vein
yellowing disease
Nicotiana Leaf thickening,
benthamiana chlorosis, curling
OsSWEET11, OsSWEET14 (rice bacterial blight
susceptibility genes)
OsERF922 (ethylene responsive factor
transcription factor)
elF(iso)4E (elF transcription factor)
CLCuD IR and Rep regions
elF4E (eukaryotic translation initiation factor
4E)
BeYDV (short intergenic region, trans acting
replication initiation protein)

Gene Editing and Crop Improvement Using CRISPR-Cas9 System. Leena Arora, Alka Narula. Front Plant Sci.
2017; 8: 1932.
 Genetic editing and consumption
Ethical aspects
➢ Do gene-edited foods deserve looser regulation than
transgenic foods?

Traditional
Transgenic
mutagenesis and Gene editing
organisms
hybridization
TRANSGENIC ORGANISMS VS. GENE EDITING

GMO Gene editing

• They contain foreign • They contain small

genes randomly alterations in existing
introduced into the genes that give the
genome that produce organism a beneficial
new proteins in the trait by tweaking the
organisms, giving it a levels of a protein
beneficial trait that it that was already in
did not have before the body
 https://www.nature.com/articles/d
41586-018-05814-6

• In the European Union, the Court of Justice (CJEU) ruled on July 25, 2018 that
gene-edited crops must be subject to the same strict regulations as conventional
genetically modified organisms.
https://ec.europa.eu/info/sites/info/files/201
8_11_gcsa_statement_gene_editing_1.pdf

➢ Safety:
What is important, the product or
the method?

The changes introduced by random mutagenesis are often more dramatic than
those that result from gene editing techniques, and include not only numerous
point mutations, but also significant deletions and rearrangements of genome
fragments.

The resulting mutant organisms require a lengthy analysis of the characteristics

of the organisms to identify the few mutants that possess a novel desirable
characteristic and do not exhibit unwanted characteristics. Despite this lengthy
selection process, the end products ultimately selected are likely to have
additional mutations beyond those that result in the desired trait.
• Unwanted effects will occur less frequently in gene-edited products -
potentially safer than traditional random mutagenesis products.

• The characteristics of the final product must be examined regardless

of the underlying technique used.

• The safety of an organism is determined by multiple factors such as the

specific characteristics of the organism, the environment in which it is
grown, the agricultural practices used, and exposure to humans and
animals rather than the technique used to produce it.

• The current approach does not adequately respect the motivation

behind the precautionary principle of ensuring product safety.

• Situations may arise where two products are identical, but due to the
different methods used in their production, they would have to meet
completely different regulatory requirements.
https://www.nature.com/articles/d41586-019-02162-x
❑GENE DRIVE

Later called gene drive

They manage to eradicate an entire Gene drive is used in
population of the malaria- mammals for the first time
transmitting mosquito (Anopheles bioRxiv 362558; doi: https://doi.org/10.1101/362558
gambiae) in captivity Super-Mendelian inheritance mediated by CRISPR/Cas9
in the female mouse germline
Nat Biotechnol. 2018 Dec;36(11):1062-1066 Grunwald HA, et al.
A CRISPR-Cas9 gene drive targeting doublesex causes complete
population suppression in caged Anopheles gambiae mosquitoes.
Kyrou K, et al.
Gene drive
Ethical issues
➢ Unpredictable consequences for the ecosystem

➢ Appearance of other pests

➢ Imbalance in the food chain ...

➢ Possible spread of mutated guide RNAs

➢ Dual-use technology