seminar on crispr
TRANSCRIPT
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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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