1. CRISPR is a bacterial immune system that provides immunity against viruses. It works by matching DNA sequences from viruses to DNA spacers and cleaving invading DNA. 2. The type II CRISPR system is the most well studied and requires only three components - Cas9 protein, CRISPR RNA (crRNA), and trans-activating CRISPR RNA (tracrRNA) - to function. Combining crRNA and tracrRNA into a single RNA molecule called sgRNA was crucial for developing the CRISPR technique. 3. CRISPR technology has become a powerful genome editing tool due to its simplicity, high efficiency, low cost, and ease of use. It allows targeted

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WELCOME
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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. 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.
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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
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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
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7. A generalised CRISPR-Cas locus
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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
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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
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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.
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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.
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Cas9 structure
The target recognition and nuclease activity of Cas 9 are independent
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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).
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15. Out of all the CRISPR systems ,type-ii is most well studied and most
simple requiring only three components.
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18. 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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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
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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
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28. What makes CRISPR system the ideal genome engineering
technology
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29. The real secret for popularity of CRISPR/Cas9 system
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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.
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32. Tartgeted mutation/correction using paired Cas9 nickase by HDR
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33. CRISPRi- CRISPR interference
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35. Paired dCas9 to produce double strand break with less off-targets6/21/2017 35

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.
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38. 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

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
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42. 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 fusobacteria
Leptotrichia shahii can target RNA.By manipulating this system
we can develop RNA editing and tracking tools.

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
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Solutions
Off target indels
Limited choice of PAM sequences

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.
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47. Future Prospects
Realizing the promise of gene therapy
Development of personalized therapeutics
Presenting the new face of GE
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