genome editing with crispr-cas9

Clustered regularly interspaced short palindromic repeats

(CRISPRs) and CRISPR-associated (Cas) proteins

CRISPR/Cas9 System

LOPAMUDRA NAYAK04ABT/14

What is Gene/Genome Editing?

• A process whereby researchers can introduce a modification into an endogenous gene

• Disruption, Insertion, Replacement at a locus in the genome

– Control gene expression

– Create SNP

– Create Reporter fusions while maintaining endogenous gene regulation

What is CRISPR (CRISPR-Cas; CRISPR-Cas9)?

• Mechanism of adaptive immunity inbacteria and archaea

• Evolved to adapt and defend againstforeign genetic material (e.g. phage)

• Several different types of CRISPRpathways in bacteria and archaea

• Type II: CRISPR-Cas9. Creates a double-strand break in the targeted DNA

Two major repair pathways of DSBs

THE CRISPR/CAS9 TEAM

crRNA + tracrRNA

Applications using CRISPR/Cas9 system

Non-protein Coding Gene disruption

Applications using CRISPR/Cas9 system

Conditional knockout- For essential genes or tissue-specific study inserting LoxP sitesaround the exon to be knocked-out

Large chromosomal deletions- using two sgRNAs

to induce DSBs at sites that flankthe region of interest

Nucl. Acids Res. June 6 (2013)

Methods of Transfection

NON VIRAL DNA DELIVERY

1.CHEMICAL METHODS 2.PHYSICAL METHOD

LIPID MEDIATED TRANSFECTION

DNA

LIPOSOME

Pro- High efficiencyCon- Optimization required

CALCIUM PHOSPHATE TRANSFECTION

Pro- InexpensiveCon- Cytotoxic to many cell types

Electroporation

Pro- Fast; No limitation of DNA sizeCon- Substantial cell death

VIRAL DNA DELIVERY METHOD

-Modified replication deficient viruses

Lentivirus

Adenovirus

Adenovirus Associated Virus(AAV)

COMPONENTS OF CRISPR/Cas9 SYSTEM

• The CRISPR /Cas9 system requires severalcomponents,

the nuclease, the guide RNA,and usually a selectable marker to enable plants

containing the components to be identified.• These components can be delivered in a similar

way to the introduction of genes in geneticmodification.

• Components using Agrobacterium-mediatedtransformation is a preferred delivery methodbecause it leads to introduction of single copiesof the genes in 50% of cases.

Sanger sequencing

197 nt deletion

1 nt insertion

68

(A)

(B)

Why is there a “CRISPR Craze”?

• Cas9 can be programmed toperform gene editing in“mammalian cells”.

• Changing a short RNA sequencecan easily target to a different sitein the genome

• Simpler and easier than othergenome editing technologies (ZFN,TALENs)

• “unprecedented efficiency andstunning ease of use”

~ Science (2014) 344(6185):707-8

• Gene therapy is back!

SCIENCE VOL 341 23 AUGUST 2013

2 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5

The patent war intensied

CASE STUDY

Background: The CRISPR/Cas9 system provides bacteria and archaea with

molecular immunity against invading phages and conjugative plasmids. Recently,

CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic

species.

Results:

The CRISPR/Cas9 system could be used in plants to confer molecular immunity

against DNA viruses. The delivery of sgRNAs specific for coding and non-coding

sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana

plants stably overexpressing the Cas9 endonuclease, and subsequently

challenge these plants with TYLCV.

Data demonstrated that the CRISPR/Cas9 system targeted TYLCV for

degradation and introduced mutations at the target sequences.

All tested sgRNAs exhibit interference activity, but those targeting the stem-loop

sequence within the TYLCV origin of replication in the intergenic region (IR) are

the most effective.

N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced

accumulation of viral DNA, abolishing or significantly attenuating symptoms of

infection. Moreover, this system could simultaneously target multiple DNA

viruses.

Conclusions: These data establish the efficacy of the CRISPR/Cas9 system for viral

interference in plants, thereby extending the utility of this technology and

An anti-browning mushroom developed by

plant pathologist Yinong Yang using CRISPR-Cas9 gene-editing technology will

have a longer shelf life and resist blemishes

from handling and mechanical harvesting.

USDA has ruled that the mushroom is not

subject to the agency's regulatory process

for GMOs.

DNA-free CRISPR

Conventionally, researchers get CRISPR/Cas9 working in aplant cell by first shuttling in the gene that codes for the Cas9enzyme. The gene is introduced on a plasmid — a circularpacket of DNA — which is usually carried into a plant by thebacterial pest Agrobacterium tumefaciens. As aresult, Agrobacterium DNA can end up in the plant’s genome.Even if the pest is not used, fragments of the Cas9 gene maythemselves be incorporated into the plant's genome.

Avoiding gene-shuttling altogether. Assembling the Cas9enzyme together with its guide RNA sequences (which theenzyme requires to find its target) outside the plant, and usesolvents to get the resulting protein complex into the plant.The technique works efficiently to knock out selected genes intobacco plants, rice, lettuce etc. thus reported in NatureBiotechnology.

conclusion

The CRISPR/Cas 9 technique is one of a numberof gene‐editing tools.

Many favour the CRISPR/Cas9 techniquebecause of its high degree of flexibility andaccuracy in cutting and pasting DNA.

One of the reasons for its popularity is that itmakes it possible to carry out geneticengineering on an unprecedented scale at avery low cost.

How it differs from previous genetic engineeringtechniques is that it allows for theintroduction or removal of more than onegene at a time. Cas9 system is efficient , site-specific, can be “multiplexed”.

This makes it possible to manipulate manydifferent genes in a cell line, plant or animalvery quickly, reducing the process from takinga number of years to a matter of weeks.

It is also different in that it is notspecies‐specific, so can be used on organismspreviously resistant to genetic engineering.

References

• Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (August 2012). "A programmable dualRNAguided DNA endonuclease in adaptive bacterial immunity". Science 337 (6096): 816–821. Bibcode:2012Sci...337..816J. doi:10.1126/science.1225829. PMID 22745249.

• Upadhyay, S.K., Kumar, J., Alok, A. & Tuli, R. RNA guided genome editing for target gene mutations in wheat. G3 (Bethesda) 3, 2233–2238 (2013).

• Feng, Z. et al. Efficient genome editing in plants using a CRISPR/Cassystem. Cell Res. 23, 1229–1232 (2013).

• Miao, J. et al. Targeted mutagenesis in rice using CRISPRCas system. Cell Res. 23, 1233–1236 (2013).

• Jiang, W. et al. Demonstration of CRISPR/Cas9/sgRNAmediatedtargeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic acids Res. 41, e188 (2013).