CRISPR cas : A new genome editing tool
-: Major Guide :-
Dr. Rukam S. Tomar
Assistant Professor
Dept. of Biotechnology
JAU, Junagadh
-: Minor Guide :-
Dr. M. K. Mandavia
Professor and Head
Dept. of Biochemistry
JAU, Junagadh
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-: Speaker :-
Abhay A. Pala
Regd. No.: J4-01390-2014
M.Sc. (Plant Mol. Biology & Biotechnology)
Dept. of Biotechnology
JAU, Junagadh

Content…
1. Introduction
2. History
3. CRISPR in bacteria
4. Classification of CRISPR
5. General structure of cas9 protein
6. Mechanism of CRISPR cas9
7. Applications
8. Data base of CRISPR
9. Case studies
10. Conclusion
11. Future aspects

Introduction…
• Genome editing, or genome editing with engineered
nucleases (GEEN) is a type of genetic engineering in which DNA is
inserted, replaced, or removed from a genome using artificially
engineered nucleases, or "molecular scissors”.
• The nucleases create specific double-strand breaks (DSBs) at
desired locations in the genome and harness the cell’s endogenous
mechanisms to repair the induced break by natural processes
of homologous recombination (HR) and non-homologous end-
joining (NHEJ).
3

Why genome editing?
 To understand the function of a gene or a protein, one interferes with
it in a sequence-specific way and monitors its effects on the organism.
 In some organisms, it is difficult or impossible to perform site-specific
mutagenesis, and therefore more indirect methods must be used, such as
silencing the gene of interest by short RNA interference (siRNA).
 But sometime gene disruption by siRNA can be variable or incomplete.
 Nucleases such as CRISPR can cut any targeted position in the genome and
introduce a modification of the endogenous sequences for genes that are
impossible to specifically target using conventional RNAi.
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Comparison between traditional and modern genome editing
technologies
Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-
ray or fast neutron
radiation)
Biological (ZFNs, TALENs
or CRISPR/ Cas)
Biological- Transgenics
(e.g., Agro or gene gun)
Characteristics
of genetic
variation
Substitution and Deletion Deletion and
chromosomal mutation
Substitution and Deletion
and insertion
Insertions
Loss of function Loss of function Loss of function and gain
of function
Loss of function and gain
of function
Advantages Unnecessary of knowing gene
function or sequences
Unnecessary of knowing
gene function or
sequences
Gene specific mutation Insertion of genes of
known functions into host
plant genome
Easy production of random
mutation
Easy production of
random mutation
Efficient production of
desirable mutation
Efficient creation of plants
with desirable traits
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Disadvantages Inefficient screening of
desirable traits
Inefficient screening
of desirable traits
Necessity of knowing
gene function and
sequences
Necessity of knowing
gene function and
sequences
Non specific mutation Non specific
mutation
Prerequisite of
efficient genetic
transformation
Prerequisite of
efficient genetic
transformation
Other features Non transgenic process
and traits
Non transgenic
process and traits
Transgenic process
but non transgenic
traits
Transgenic process
and traits
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Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-
ray or fast neutron
radiation)
Biological (ZFNs, TALENs
or CRISPR/ Cas)
Biological- Transgenics
(e.g., Agro or gene gun)

1987
• Researchers find CRISPR sequences in Escherichia coli, but do not
characterize their function.
2000
• CRISPR sequence are found to be common in other microbes.
2002
• Coined CRISPR name, defined signature Cas genes
2007
• First experimental evidence for CRISPR adaptive immunity
2013
• First demonstration of Cas9 genome engineering in eukaryotic cell
HISTORY
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CRISPR – Cas systems
• These are the part of the Bacterial immune system which detects
and recognize the foreign DNA and cleaves it.
1. THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
loci
2. Cas (CRISPR- associated) proteins can target and cleave invading DNA in a
sequence – specific manner.
A CRISPR array is composed of a series of repeats interspaced by
spacer sequences acquired from invading genomes.
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Components of CRISPR
1. Protospacer adjacent motif (PAM)
2. CRISPR-RNA (crRNA)
3. trans-activating crRNA (tracrRNA)
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Different CRISPR-Cas system in Bacterial Adaptive Immunity
Class 1- type I (CRISPR-Cas3) and type III (CRISPR-
Cas10)
 uses several Cas proteins and the crRNA
Class 2- type II (CRISPR-Cas9) and type V (CRISPR-
Cpf1)
 employ a large single-component Cas-9 protein in
conjunction with crRNA and tracerRNA.
11Zetsche et al., (2015)
functioning of type II CRISPR system

Different Cas proteins and their function
Protein Distribution Process Function
Cas1 Universal Spacer acquisition DNAse, not sequence specfic, can bind RNA; present in all Types
Cas2 Universal Spacer acquisition specific to U-rich regions; present in all Types
Cas3 Type I signature Target interference DNA helicase, endonuclease
Cas4 Type I, II Spacer acquisition RecB-like nuclease with exonuclease activity homologous to RecB
Cas5 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas6 Type I, III crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas7 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas8 Type I crRNA expression Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of
CASCADE
Cas9 Type II signature Target interference Large multidomain protein with McrA-HNH nuclease domain and RuvC-like
nuclease domain; necessary for interference and target cleavage
Cas10 Type III signature crRNA expression
and interference
HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE
elements 12

Action of CRISPR in bacteria
The CRISPR immune system works to protect bacteria from
repeated viral attack via three basic steps:
(1) Adaptation
(2) Production of cr RNA
(3) Targeting
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Structure of cas9 protein

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Structure of crRNA

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Versatile Nature of CRISPR Technology
20Jeffry et al., 2014

CRISPR/Cas9-based knock-out of phytoene
desaturase gene (PDS) in Populus tomentosa
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Combining crRNA and tracrRNA into sgRNA was the crucial step
for the development of CRISPR technology
22Joung et al., 2012

What makes CRISPR system the ideal genome engineering technology
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Examples of crops modified with CRISPR technology
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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

Genome editing
tool
Transformation
method
Crops Targeted genes
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General protocol for CRISPR

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RECENT ADVANCES
Discovery of new version of Cas9
Engineered Cas9 with altered PAM specificity
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Cpf1 (CRISPR from Prevotella and Francisella 1) at Broad Institute
of MIT and Harvard, Cambridge.
CRISPR-Cpf1 is a class 2 CRISPR system
Cpf1 is a CRISPR-associated two-component RNA programmable
DNA nuclease
Does not require tracerRNA and the gene is 1kb smaller
Targeted DNA is cleaved as a 5 nt staggered cut distal to a 5’ T-rich
PAM
Cpf1 exhibit robust nuclease activity in human cells
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Zetsche et al., (October 22, 2015)
New Version of Cas9:

Cpf1 makes staggered cut at 5’
distal end from the PAM
30
Organization of two CRISPR loci found in Francisella
novicida .The domain architectures of FnCas9 and FnCpf1
are compared

DNAi-Targeted DNA degradation
31Brian J. et al., 2015
 Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce
environmental release and protect intellectual property.
 Here is a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-
defined DNA.
 This enables engineered regions to be obscured when the cell enters a new environment.
 DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target.
 When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10^8

Application in Agriculture
 Can be used to create high degree of genetic variability at precise locus in the
genome of the crop plants.
 Potential tool for multiplexed reverse and forward genetic study.
 Precise transgene integration at specific loci.
 Developing biotic and abiotic resistant traits in crop plants.
 Potential tool for developing virus resistant crop varieties.
 Can be used to eradicate unwanted species like herbicide resistant weeds,
insect pest.
 Potential tool for improving polyploid crops like potato and wheat.
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Some pitfalls of this technology
 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

How to avoid off-target effects?
- Optimization of Injection conditions (less cas9/sgRNA)
- Bioinformatics : Find a sgRNA target for less off-targets
“CRISPR Design” (http://crispr.mit.edu)
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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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Case Studies
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Large chromosomal deletions and heritable small
genetic changes induced by CRISPR/Cas9 in rice
Case study 1
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Yang et al. (2014) USA

MATERIALS AND METHODS
1. Construction of plasmids expressing Cas9 and guide RNAs
-pUbi-Cas9 binary vactor which is synthesise by GeneScrept and
cloned into pENTR4
2. Transient gene expression in rice protoplasts
-Isolation and transfection of rice mesophyll protoplasts
were carried out.
3. Agrobacterium-mediated rice transformation
-Agrobacterium tumefaciens strain EHA105 by electroporation.
4. Detection of large fragment deletion by T7E1
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Structure of Binary Vector

(A) Schematic of a cluster of five diterpenoid genes
within an ~170kb region on rice chromosome
number 4.
(B) Agarose gel electrophoresis image of
the PCR products amplified from DNA.
(C) Sequences of large DNA segment deletions
induced by sgRNAs.
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Callus Line of T0 plant
(B) Callus lines containing the ~245kb
deletions identified with PCR approach
and confirmed with sequencing of
amplicons.
(C) DNA sequence changes in the
representative plants generated from
three callus lines (#16, 17 and 21) with
the Cas9/ sgRNA induce large deletions
(dashed lines) in one chromosome and
small nucleotide changes (dashed lines for
deletions and lower case letters for
insertions) in the homologous
chromosome.

Targeted genome modifications in soybean with
CRISPR/Cas9
Jacobs et al.(2015) USA
Case study-2

MATERIALS AND METHODS
• Plant material
• Plasmid construction of cas9 and sgRNA
• Hairy root transformation of soybean by A. rhizogenes (strain K599)
• GFP imaging via Olympus MVX10 microscope with a GFP filter
• sequencing and analysis
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Schematic showing the targeted GFP sequences.

(B)C9 + GFP 5' target events
• Wild-type sequences are in green, deletions are shown as
dashes, and SNPs are shown in orange.

(C) C9 + GFP 3' target events
• Wild-type sequences are in green, deletions are shown as dashes,
and SNPs are shown in orange.

Modification efficiency for hairy root events
1. 01gDDM1- 78-80% indel frequency
2. 11gDDM1- 87-90% indel frequency
3. 01gDDM1 +11gDDM1 – 21-23% indel frequency

Conclusion…
Genome editing tools provide new strategies for genetic manipulation in plants and are likely
to assist in engineering desired plant traits by modifying endogenous genes.
Genome editing technology will have a major impact in applied crop improvement and
commercial product development .
CRISPR will no doubt be revolutionized by virtue of being able to make targeted DNA
sequence modifications rather than random changes.
In gene modification, these targetable nucleases have potential applications to become
alternatives to standard breeding methods to identify novel traits in economically important
plants and more valuable in biotechnology as modifying specific site rather than whole
gene.
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