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  1. 1. 1 WELCOME 6/21/2017
  2. 2. 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 , Neri6/21/2017 2
  3. 3. In 1987 Ishino et al. first described a pattern of short palindromic repeats of DNA interspaced with short,non repititive “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. 6/21/2017
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  5. 5. 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 6/21/2017 5
  6. 6. CRISPR as bacterial immune system against bacteriophagy The research was carried at by researchers in DANISCO.Inc (acquired by DuPont at 2011) Science 2007 6/21/2017 6
  7. 7. A generalised CRISPR-Cas locus 6/21/2017 7
  8. 8. 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 6/21/2017 8
  9. 9. 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 bp DNA 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 6/21/2017 9
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  11. 11. 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. 6/21/2017 11
  12. 12. 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. 12 Cas9 structure The target recognition and nuclease activity of Cas 9 are independent 6/21/2017
  13. 13. 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). 6/21/2017 13
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  15. 15. Out of all the CRISPR systems ,type-ii is most well studied and most simple requiring only three components. 6/21/2017 15
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  18. 18. Combining crRNA and tarcerRNA into sgRNA was the crucial step for the development of CRISPR technology 6/21/2017 18
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  21. 21. 21 General protocol for CRISPR 6/21/2017
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  23. 23. 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 mutagenesis Gene editing Shaohui et al., 2015 Yupeng et al., 2015 Harrison et al., 2014 6/21/2017 23
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  25. 25. 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 256/21/2017
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  28. 28. What makes CRISPR system the ideal genome engineering technology 6/21/2017 28
  29. 29. The real secret for popularity of CRISPR/Cas9 system 6/21/2017 29
  30. 30. Cas9 Nuclease can be engineered for a variety of applications.ex-In Cas9 nickase one nuclease domain has been mutated and made non- functional. 6/21/2017 30
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  32. 32. Tartgeted mutation/correction using paired Cas9 nickase by HDR 6/21/2017 32
  33. 33. CRISPRi- CRISPR interference 6/21/2017 33
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  35. 35. Paired dCas9 to produce double strand break with less off-targets6/21/2017 35
  36. 36. 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. 6/21/2017 36
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  38. 38. Photoactivatable CRISPR-Cas9 for Optogenetic Genome Editing 6/21/2017 38 Based on a recently developed photoinducible dimerization system named Magnets
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  40. 40. 6/21/2017 40 The Mutagenic Chain Reaction: A method for converting heterozygous mutation to homozygous mutations
  41. 41. Construction of gene drive Gene Drive is a technique that promotes the inheritance of a particular gene to increase its prevalance in a population 6/21/2017 41
  42. 42. Discovery of CRISPR/Cpf1 Discovery of CRISPR C2c2 system Engineered Cas9 with altered PAM specificity Engineered Cas9 for better specificity(eSpCas9,SpCas9-HF1) 6/21/2017 42 RECENT ADVANCES
  43. 43. 6/21/2017 43  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
  44. 44. 6/21/2017 44 In 2016 researchers demonstrated CRISPR from fusobacteria Leptotrichia shahii can target RNA.By manipulating this system we can develop RNA editing and tracking tools.
  45. 45. 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 HDR 6/21/2017 45 Solutions Off target indels Limited choice of PAM sequences
  46. 46. 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. 466/21/2017
  47. 47. Future Prospects Realizing the promise of gene therapy Development of personalized therapeutics Presenting the new face of GE 6/21/2017 47
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