CRISPR cas, a potential tool for targeted genome modification in crops.

CRISPR –cas
A Potential Tool for Genome Modification
MONOJ SUTRADHAR
PALB 3243
Sr. M.sc, Plant Biotechnology
UAS, GKVK,Bangalore
Department of Plant Biotechnology,UAS,GKVK,Bangalore
25 October 2014 1

Contents
 Introduction
 History
 Mechanism overview
 Types of CRISPR-cas system
 Cas9 nuclease
 Comparisons among different kinds of nucleases
 Case study
 Conclusion
25 October 2014
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CRISPR
CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci
containing short repetitions of base sequences which separated by short "spacer
DNA" from previous exposures to a virus or phage.
• It represents a family of DNA repeats in most archaeal (~90%) and bacterial
(~40%) genomes provides acquired immunity against viruses and phages.
(Barrangou et al.,2010)
25 October 2014 3

The size of CRISPR repeats and spacers varies between 23 to 47
base pairs (bp) and 21 to 72 bp, respectively. Generally, CRISPR
repeat sequences are highly conserved within a given CRISPR
locus.
25 October 2014
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• The CRISPR leader, defined as a low-complexity, A/T-rich,
noncoding sequence, located immediately upstream of the first
repeat, likely acts as a promoter for the transcription of the
repeat-spacer array into a CRISPR transcript, the pre-crRNA.
The full-length pre-crRNA is subsequently processed into specific
small RNA molecules that correspond to a spacer flanked by two
partial repeats.
25 October 2014 Department of Plant Biotechnology,UAS,GKVK,Bangalore 5

HISTORY
6
Zhang et al.,2014
Department of Plant Biotechnology,UAS,GKVK,Bangalore 25 October 2014

Types of CRISPR CAS system
 There are three types of CRISPR-Cas systems, which vary in their
specific target and mechanism of action.
 Type I systems cleave and degrade DNA,
 Type II systems cleave DNA ,
 Type III systems cleave DNA or RNA .
Type I and II systems require two principal factors to effectively
target DNA:
 (i) complementarity between the CRISPR RNA spacer and the
target “protospacer” sequence.
(ii) a protospacer-adjacent motif (PAM) specific to each CRISPR-Cas
system flanking the protospacer.
Department of Plant Biotechnology,UAS,GKVK,Bangalore 7 25 October 2014

• Effective targeting can occur even for multiple mismatches
between the CRISPR RNA and the protospacer, although
mismatches within the “seed” region flanking the PAM are
more disruptive .
Similar factors are required for DNA-targeting by type III
systems, where these systems evaluate base pairing
between the target sequence and the region flanking the
protospacer.
Department of Plant Biotechnology,UAS,GKVK,Bangalore 25 O8ctober 2014

Mechanism of CRISPR-cas system
Mechanistically, although defense is spacer-encoded, the information that lies within the
CRISPR repeat- spacer array becomes available to the Cas machinery through transcription
11

 CRISPR-Cas systems are RNA-directed adaptive immune systems in many
bacteria and most archaea that recognize nucleic acids of invading plasmids
and viruses.
25 October 2014
Department of Plant Biotechnology,UAS,GKVK,Bangalore 12

Recognition is directed by CRISPR RNAs that are processed from transcribed
arrays of alternating target specific“spacer”sequences and identical “repeat”
sequences.

The spacer region of each CRISPR RNA base pairs with complementary nucleic acids,
driving cleavage or degradation by the Cas proteins within minutes of invasion.

15
Barrangou et al.,2012

DNA repair
Zhang et al.,2014

CRISPR interference

Terns et al.,2014
RNA programmable gene knockdown by Type iii-B Cmr CRISPR-cas system.

The Cmr effector complex(blue) comprises six Cmr proteins and a
crRNA.
The guide region of crRNA base-pairs with the homologous sequence
in the mRNA target and the target RNA is cleaved by the complex.
Applications of Cmr system include RNA-directed gene knockdown to
investigate gene function or to facilitate metabolllic engineering.

In bacterial type 2 CRISPR cas
system, the site specificity is
defined by complementery base
pairing of a small CRISPR
RNA(crRNA
After annealing to a
transactiviting CRISPR
RNA(tracrRNA) the crRNA directly
guides the cas9 endonuclease to
cleave the targeted DNA sequence.
The crRNA–transcr- RNA
heteroduplex could be replaced by
one chimeric RNA (so-called guide
RNA (gRNA)) and the gRNA could
be programmed to target specific
sites.
Jinek et al.,2012

25 October 2014 Department of Plant Biotechnology,UAS,GKVK,Bangalore 24 Joung et al.,2012

Comparision between sgRNA and
crRNA-tracrRNA hetero duplex
•Advantages
• Flexible targeting
• Sequence specific
• Transferable(codon optimized,NLS)
• Efficient
• Precise cleavage
• Affordable
• Quick
• Multiplex guides
• Multiplex orthogonal system
Caveats
 Cas9 is a large protein
 PAM – dependent design limitations
 Off –target cleavage

In Humans,Zebrafish,Drossophilla,Mice,rats genome editing has been
achived by combining Streptococcus pyogenes cas9 and a synthetic
single guide RNA(sgRNA) consisting of CRISPR RNA(crRNA) and
tracrRNA.
(Joung et al.,2013),(Gartz et al.,2013), (Susan et al.,2014)
The CRISPR/cas9 system has also been used in model plants like
Nicotiana benthamiana, Arabidopsis thaliana and crops like
wheat,rice,sorghum by transient or stable transformation.
(Cong et al.,2013),(Jiang et al.,2013),

Other nucleases
Sequence-specific nucleases increase the efficiency of gene targeting.
Among them, zinc-finger nucleases (ZFNs) and transcription activator-like
effector nucleases (TALENs) are the two most commonly used
sequence-specific chimeric proteins.

Gao et al.,2014

Once the ZFN or TALEN constructs are introduced into and expressed
in cells, their programmable DNA-binding domains can specifically
bind to a corresponding sequence and guide the chimeric nuclease
(e.g. FokI nuclease) to make a specific DNA strand cleavage.
In general, single zinc-finger motif specifically recognizes 3 bp, and
engineered zinc-finger with tandem repeats can recognize up to 9–36
bp. However, it is quite tedious and time-consuming to screen and
identify a desirable ZFN (Pabo et al., 2001).

CASE STUDY

Introduction
 The bacterial Type II cluster regularly interspaced short palindromic
repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool
for genome editing in microbial and animal systems as well as in plants.
 Three guide RNAs (gRNAs) with a 20–22-nt seed region were
designed to pair with distinct rice genomic sites which are
followed by the PAM.

The secondary structure of gRNA mimics the crRNA–transcrRNA
heteroduplex that binds to Cas9.
The 5′-end of gRNA (gRNA seed) pair with one strand of targeted
DNA. The scaffold of gRNA is labeled with dark-red cycles.
A PAM motif (N-G-G) is located adjacent to the DNA– gRNA pairing
region in the complementary strand of targeted DNA.
The Cas9 nuclease would cleave both strands of DNA at a
conserved position which is 3 bp to the PAM motif.

pRGE vectors for transient expression.

A DNA dependent RNA polymerase III promoter and terminator are used to
control the transcription of engineered gRNA.
 Rice pol III promoters(snoRNA U3 and U6 promoters) were isolated
to make pRGE3 and pRGE6 vectors.
 Plant DNA dependent RNA polymerase II(pol II) promoter and
terminator are used to control the expression of a chimeric cas9
nuclease
 hspCas9 encodes a human codon optimized Cas9 nuclease which
includes a nuclear localization signal(NLS) and a FLAG tag. Amp
represents an ampicillin resistance gene.

The designed oligonucleotides duplex can be inserted into Bsal sites in pRGE
vectors and fused with gRNA scaffold to construct engineered gRNA.
 The sequence with red colour will be replaced by designed DNA
sequence encoding gRNA.
 Italic lower case letter indicates overhang sequence after Bsal
digestion

Design of gRNAs to target three specific sites of OsMPK5.
The targeted sites by engineered gRNA(PS1-3) are shown as PS1,PS2
and PS3.

The rectangles represent exons of which the black ones indicate
the OsMPK5 coding region.
• PS1 contains a Kpnl site and PS3 contains a Sacl site.
F-256 and R-611 indicate the position of primers used to amplify
genomic fragment of OsMPK5.

• Base pairing between the seed region of engineered gRNAs
and the targeted sites in the OsMPK5 gene.
PS1-gRNA was paired with the coding strand of OsMPK5
whereas PS2 and PS3 gRNA were paired with the
template strand of OsMPK5.
The predicted gRNA-Cas9 cutting position was indicated with the
scissor symbol. The PAM was shown in red colour

Detection of Genome editing and targeted mutations at the PS1
and PS3 sites in the OsMPK5 locus by RE-PCR.

Results
 The engineered gRNAs were shown to direct the Cas9 nuclease for precise
cleavage at the desired sites and introduce mutation (insertion or deletion)
by error-prone non-homologous end joining DNA repairing with 3–8%
efficiency.
Further analysis suggests that mismatch position between gRNA
seed and target DNA is an important determinant of the gRNA–Cas9
targeting specificity, and specific gRNAs could be designed to target
more than 90% of rice genes.

CONCLUSION
The CRISPR cas utilize guide RNAs to effectively recognize and target foreign
DNA and RNA for destruction.
RNA guided recognition make this immune system highly and rapidly
adaptable to diverse targets(recognizing new targets require guide RNA
sequence which can be obtained directly from invader).
Flexible and accessible tool for multiple application like genome editing and
modulation of gene expression.
Notably understanding of the multiple CRISPR cas system is far from
complete and additional tools and applications are yet to come from fertile
research.
Department of Plant Biotechnology, UAS,GKVK,Bangalore 48 25 October 2014

CRISPR cas, a potential tool for targeted genome modification in crops.

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