“CRISPR/Cas9 in zebrafish: an efficient

combination for human

genetic diseases modeling”
 Outline
 Introduction
 Zebrafish as a Disease Model
 Genome Editing tools for Zebrafish (CRISPR/Cas9, ZFNs
and TALENs)
 CRISPR/Cas9 from Adaptive Immunity to Genome
Engineering
 Successful Gene Functional Studies
 Multiplex Biallelic Genome Editing
 Future Developments and Promises
 Conclusion
 Introduction
 Next generation sequence technologies like WES,
WGS helps in the genetic variant analysis.

 But these technologies are insufficient in

distinguishing the disease causing variants from
potentially functional variants.
 Genome sequencing of zebrafish revealed that 70% of
the human genes have homologoes in zebrafish.

 CISPR/Cas9 can be used to induce mutations in the

genome of zebrafish (a disease model).

 The mutated genes can be used for studying the

human pathogenesis.
Zebrafish as a Disease Model
Zebrafish as a Model for Studying the Human
Pathogenesis

 Mouse models have been used for studying the human

genetic diseases.

 Mouse models have few challenges.

 Their number of progeny is very small.

 High cost is required to utilize mouse in high-

throughput analysis.
 Many features like external fertilization, rapid development
and transparency of its larvae make it an ideal model.

 70% of human genes have functional homologes in zebrafish.

 For studying the human diseases, mutations in zebrafish

genome can be introduced by either random mutagenesis or by
CRISPR/Cas9/ZFNs/ TALENs.

 Chemical screening of mutant phenotypes is easy in zebrafish.

 Drawbacks of Random Mutagenesis
 Random mutagenesis generates heterozygous mutants.

 It also produces recessive inherited phenotypes that

are difficult to detect.

 Every gene can not be mutated by using random

mutagenesis.

 These problems of random mutagenesis can be

overcome by CRISPR/Cas9.
 Genome Editing Tools for Zebrafish

 CRISPR/Cas9 System

 Zinc Finger Nucleases (ZFNs)

 Transcription activator-like effector nucleases

(TALENs)
CRISPR/Cas9 as a part of adaptive immunity

 CRISPR sequence was first discovered in

prokaryotes as a part of natural defense mechanism.

 CRISPR consists of repeated nucleotides that are

interrupted by spacer sequences.

 CRISPR associated (cas) sequence is present

adjacent to CRISPR loci.
 How CRISPR Works?

 Identification of previously encountered infection

 crRNA Processing

 crRNA forms complex with Cas9.

 Binding of crRNA to target sequence.

 Cas 9 activation and breakage of dsDNA.

 Engineered CRISPR/Cas9 system
 Later, it was found that tracrRNA hybridized to crRNA
during its maturation.
 Two-RNA structure is now replaced with engineered
single guide RNA (sgRNA).
 sgRNA guides Cas9 to target sequence which then induce
cuts in DNA.
 Engineered CRISPR/Cas9 system is different from other
systems in that it shows better nuclear expression and
localization.
• Cas9 and sgRNA, incorporated into the fish genome
downstream of tissue-specific promoters. DSBs are
induced and genome editing can be accomplished.
Schematic illustration of the components of engineered CRISPR-
Cas9 systems.
 ZFNs for Genome Editing
 Artificially created Nucleases.

 ZFNs consist of DNA binding domains and DNA

cleavage domains.

 DNA cleavage domain induce DSBs in dimeric form.

 Errors in NHEJ repair process induce mutations in

DNA.
A pair of ZFNs bound to DNA
 TALENs as a Tool for Genome Engineering

 Engineered Nucleases.

 Developed by fusing sequence specific DNA

binding domain of TALEs with non-specific
Nucleases.

 Induce DSBs in the target site of DNA.

Cutting of DNA by TALENs
 Most Amenable Approach for Mutagenesis

 Compared to ZFNs and TALENs, CRISPR/Cas9 is the

most preferred for mutagenesis.
 Easy programmability of DNA binding domains of
sgRNA.
 Various softwares are available for designing of
sgRNA with minimum off-target effects (CRISPR
MultiTargeter, CRISPRdirect, CHOPCHOP).
 Successful Gene Function Studies
 Successful mutations have induced in zebrafish genome by
CRISPR/Cas9.

 Effect of MMP21 knock-out (responsible for heterotaxy in

humans)

Cardiac looping defects and defects in Notch signaling

in zebrafish embryos.

 Recent high throughput studies involving CRISPR/Cas9 revealed

that mutations were successfully induced in 99% of the genes
tested and 28% of them are transferred to next generations.
 Multiplex biallelic genome editing
• Multiplex biallelic genome editing can also be
achieved simultaneously in zebrafish model by using
CRISPR/Cas9.

• Multiple phenotypes caused by mutations in multiple

genes can be studied in the same clutch of fish.
Human Genetic diseases studied in Zebrafish
 Future development and promises
• Promising future for studying human genetic
diseases.
• Candidate disease genes are being identified
continuously but their functions still need to be
determined.
• Zebrafish is an ideal biological system in this case
particularly considering its similarity to human
biology.
• On-target modifications are quite accurate for
CRISPR/Cas9 but the off-target modifications have
not been yet performed for CRISPR/Cas9 in the
zebrafish.

• Unidentified off-target modifications in the fish

genome may cause false-positive annotation of the
gene functions.
 Conclusion
NGS have identified new genetic variants whose functions
must be studied in suitable model like zebrafish.
Understanding of diseases is necessary to develop
therapeutic approaches. The recent three years has
witnessed that CRISPR/Cas9 has created in our ability to
perform targeted gene perturbations in zebrafish, with high
levels of on-target efficiency and relatively low off-target
modifications.