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WELCOME

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CRISPR FOR GENOME EDITING

Submitted to : Dr. Vikas Sharma Submitted by : Man Mohan soni

NB-2013-15-BIV

BTC -417

Department of Biotechnology

College of Horticulture and Forestery , Neri 6/21/2017 2

In 1987 Ishino et al. first described a pattern of short palindromic repeats of DNA interspaced with short,nonrepititive “spacers” of DNA in E.coli bacteria

INTRODUCTION AND HISTORY OF CRISPR

With time more such patterns were observed in other bacteria and archaea and in 2002 Jansen et al. named the pattern CRISPR,short for “clustered regularly interspaced short palindromic repeats” and also documented the existence of a number of crispr-associated genes(named the cas family)adjacent to these repeats.

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2010- Garneau et al. show that the CRISPR/Cas system can acquire new spacers from foreign DNA

2007- Barrangou et al. showed that CRISPR mediated by Cas proteins,provides bacterial immunity against viruses by matching DNA in spacer sequences with DNA from virus

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CRISPR as bacterial immune system

against bacteriophagy

The research was carried at by researchers in DANISCO.Inc

(acquired by DuPont at 2011)

Science 2007

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A generalised CRISPR-Cas locus6/21/2017 7

The new classification of CRISPR-Cas loci has been divided into two classes,five types, and 16 sub-types based on analysis of signature protein families and features of the archtitecture of Cas loci.

Types of crispr-cas system

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SOME TERMS

• 1) Protospacer- sequence present in virus or plasmid,adjacent to PAM,recognized by cas proteins as their target.

• 2)Spacer- Novel sequences(protospacer) acquired by bacteria and present between repeats

• 3)PAM-protospacer adjacent motif(PAM) is a 2-6 bpDNA sequence immediately following the dna sequence targeted by cas nucleases.PAM is a component of the invading virus or plasmid,but is not a component of the bacterial CRISPR locus

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TERMS(cont.)

• 4) pre crRNA-the single RNA obtained by trancription of repeats and spacers.

• 5)Trans activating crRNA(tracr RNA)- It is a small trans-encoded RNA found in type-ii CRISPR system. The 5-prime end of tracr-RNA sequence has has homology with repeats present in CRISPR locus,thus tracr-RNA and pre crRNA can make RNA duplex into the region of homology.

• 6)Cas9- An RNA directed DNA endonuclease.

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Responsible for binding guide RNA

Crucial for initiating cleavage activity upon binding of target DNA

The PAM-Interacting domain confers PAM specificity

The HNH and RuvC domains are nuclease domains that cut single-stranded DNA.

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Cas9 structure

The target recognition and nuclease activity of Cas 9 are independent6/21/2017

CRISPR–Cas systems function in three general steps

1) Adaptation or immunization (involving the acquisition of spacers

2) Biogenesis of CRISPR RNA (crRNA; encoded by the repeat–spacer regions)

3) interference (cleavage of invading nucleic acid).

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Out of all the CRISPR systems ,type-ii is most well studied and most simple requiring only three components.

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Combining crRNA and tarcerRNA into sgRNA was the crucial step

for the development of CRISPR technology

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General protocol for CRISPR

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Examples of crops modified with CRISPR

technology

CROPS DESCRIPTION REFERNCES

Corn Targeted mutagenesis Liang et al. 2014

Rice Targeted mutagenesis Belhaj et al. 2013

Sorghum Targeted gene modification Jiang et al. 2013b

Sweet orange Targeted genome editing Jia and Wang 2014

Tobacco Targeted mutagenesis Belhaj et al. 2013

Wheat Targeted mutagenesis Upadhyay et al. 2013,

Yanpeng et al. 2014

Potato

Soybean

Targeted mutagenesisGene editing

Shaohui et al., 2015

Yupeng et al., 2015

Harrison et al., 2014

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sgRNA designing tools

Optimized CRISPR Design (Feng Zhang's Lab at MIT/BROAD,

USA)

sgRNA Scorer (George Church's Lab at Harvard, USA)

sgRNA Designer (BROAD Institute)

ChopChop web tool (George Church's Lab at Harvard, USA)

E-CRISP (Michael Boutros' lab at DKFZ, Germany)

CRISPR Finder (Wellcome Trust Sanger Institute, Hinxton, UK)

RepeatMasker (Institute for Systems Biology) to double check and

avoid selecting target sites with repeated sequences

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What makes CRISPR system the ideal genome engineering

technology

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The real secret for popularity of CRISPR/Cas9 system

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Cas9 Nuclease can be engineered for a variety of applications.ex-In Cas9 nickase one nuclease domain has been mutated and made non-functional.

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Tartgeted mutation/correction using paired Cas9 nickase by HDR

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CRISPRi- CRISPR interference

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Paired dCas9 to produce double strand break with less off-targets6/21/2017 35

EPIGENOME EDITING

Targeted manipulation of epigenetic marks(DNA methylation /unmethylation, Histone Modification by acetylation /deacetylation) could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control.

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Photoactivatable CRISPR-Cas9 for Optogenetic

Genome Editing

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Based on a recently developed photoinducible dimerization system named Magnets

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The Mutagenic Chain Reaction: A method for

converting heterozygous mutation to homozygous mutations

Construction of gene drive

Gene Drive is a technique that promotes the inheritance of a particular gene to increase its prevalance in a population

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Discovery of CRISPR/Cpf1

Discovery of CRISPR C2c2 system

Engineered Cas9 with altered PAM

specificity

Engineered Cas9 for better

specificity(eSpCas9,SpCas9-HF1)

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RECENT ADVANCES

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Cpf1 (CRISPR from Prevotella and Francisella 1) discovered at Broad Institute of MIT and Harvard, Cambridge.

Does not require tracerRNA(two component system) and the gene is 1kb smaller

Targeted DNA is cleaved as a 5 nt staggered cut distal to a 5’ T-rich PAM

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In 2016 researchers demonstrated CRISPR from fusobacteriaLeptotrichia shahii can target RNA.By manipulating this system we can develop RNA editing and tracking tools.

Some pitfalls of CRISPR

Proper selection of gRNA

Use dCas9 version of Cas9 protein

Make sure that there is no mismatch within the seed

sequences(first 12 nt adjacent to PAM)

Use smaller gRNA of 17 nt instead of 20 nt

Sequence the organism first you want to work with

Use NHEJ inhibitor in order to boost up HDR6/21/2017 45

Solutions

Off target indels

Limited choice of PAM sequences

Conclusion

CRISPR technology has emerged as a powerful and universal technology for genome engineering with wide-ranging innovative implications across biology and medicine.

This technology has proved its potential by being user friendly and has shown its practicality in ensuring health as well as food security of the future.

The tool itself do not pose a threat and we hope that the CRISPR technology will live up to its promise by being used responsibly and carefully.

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Future Prospects

Realizing the promise of gene therapy

Development of personalized therapeutics

Presenting the new face of GE

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