An efficient genome editing tool

1. The CRISPR-Cas immune system: Biology, mechanisms and applications
Devashish Rath a, Lina Amlinger b, Archana Rath c, Magnus Lundgren b, *
a Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, India
b Department of Cell and Molecular Biology, Uppsala University, SE-751 24 Uppsala, Sweden
c Department of Biotechnology, University of Mumbai, Mumbai 400098, India
Presented by
Ashish Kumar Shukla
M.S. (Pharm)
Reg. No. BT/295
Dept. of Biotechnology
National Institute of Pharmaceutical Education and Research (NIPER), Hajipur
Review Article

2. CRISPR–Cas systems
These are the part of bacterial immune system which detects and recognize the
foreign DNA and cleaves it.
1. The CRISPR (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.
2. Cas (CRISPR-associated) proteins can target and cleave invading DNA in a
sequence-specific manner.
 It was first observed in Escherichia coli by Osaka University researcher Yoshizumi
Ishino in 1987.
 It represents a family of DNA repeats in most archaeal (~90%) and bacterial
(~40%) genomes provides acquired immunity against viruses and phages.

3. Spacer :- The direct repeats in a CRISPR locus are separated by short stretches of non-repetitive DNA called
spacers that are typically derived from invading plasmid or phage DNA.
Protospacers :-The nucleotide sequence of the spacer must be similar to a region in the phage genome called a
protospacer in order to recognize and subsequently block phage replication.
 The length and sequence of repeats and the length of spacers are well conserved within a CRISPR locus, but may
vary between CRISPRs in the same or different genomes.
 Repeat sequences are in the range of 21 bp to 48 bp, and spacers are between 26 bp and 72 bp.
 A conserved sequence associated with CRISPR loci called leader, located up-stream of the CRISPR with respect
to direction of transcription.

4.  1987- CRISPR sequences were first discovered in Escherichia coli. (Ishino et al., 1987)
 2002- Identification of Cas genes that are associated with DNA repeats in prokaryotes. (Jansen et al.,2002)
 2007- CRISPR provides acquired resistance against viruses in prokaryotes. (Barrangou et al., 2007)
 2012- Idea of using CRISPR- Cas9 as a genome engineering tool was published by Jennifer Doudna and
Emmanuelle Charpentier.
HISTORY

5. The CRISPR-Cas mediated defense process
can be divided into three stages:
 The first stage, adaptation, leads to insertion of new spacers
in the CRISPR locus.
 In the second stage, expression, the system gets ready for
action by expressing the Cas genes and transcribing the
CRISPR into a long precursor CRISPR RNA (pre-crRNA).
The pre-crRNA is subsequently processed into mature
crRNA by Cas proteins and accessory factors.
 In the third and last stage, interference, target nucleic acid
is recognized and destroyed by the combined action of
crRNA and Cas proteins complex.
CRISPR-Cas Defense Mechanism

6. Model of the adaptation in the Type I and III system.
 There are two types of spacer acquisition, naïve and primed.
Both require the presence of a PAM and are dependent on the
Cas1-Cas2 complex.
 The Cas1-Cas2 complex recognizes the CRISPR and likely
prepares it for spacer integration.
 Naïve spacer acquisition occurs when there is no previous
information about the target in the CRISPR.
 Primed spacer acquisition re-quires a spacer in the CRISPR
locus that matches the target DNA and the presence of Cas3
and the Cascade complex. Primed acquisition results in
insertion of more spacers from same mobile genetic element.
PAM = Protospacer Adjacent Motif.

7. Model of crRNA processing and interference.
• In Type I systems, the pre-crRNA is
processed by Cas5 or Cas6.
• DNA target interference requires
Cas3 in addition to Cascade and
crRNA.

8. • Type II systems use RNase III
and tracrRNA for crRNA
processing together with an
unknown additional factor that
perform 50 end trimming.
• Cas9 targets DNA in a crRNA-
guided manner.
• The Type III systems use Cas6
for crRNA processing, but in
addition an unknown factor
perform 30 end trimming.
• Here, the Type III Csm/Cmr
complex is drawn as targeting
DNA, but RNA may also be
targeted.

9. Hypothesis for CRISPR-Cas system evolution.
• A casposon inserts adjacent to a Cascade
operon with protein-based non-adaptive
targeting ability.
• The casposon subsequently loses genes and
the terminal inverted repeats (TIRs) expand
into a CRISPR cluster to eventually form
Type I and III CRISPR-Cas systems.
• The Type II CRISPR-Cas system development
is initiated when a transposon containing cas9
replaces the Cascade genes.

10. Application of CRISPR-Cas9
as Genome Editing Tool
1.Delivery of desire gene.
2.Editing.
3.Knockdown/activation.
4.Disease models study.
5.Biomedicine development.
6.In vitro genetic depletion.
Etc.

11.  The sgRNA directs Cas9 cleavage of the corresponding target to initiate gene editing.
 In eukaryotic cells, two main pathways repair DNA damage: Non-Homologous End Joining
(NHEJ) and Homology Directed Repair (HDR).
 NHEJ removes bases, often causing a frameshift and inactivation of the gene. HDR can be used to
make specific changes to the target region by providing a designed repair template that becomes
inserted in the damaged region.
Complex of crRNA and tracrRNA form sgRNA

12. Gene silencing using Cascade and Cas9.
 In absence of Cas3, Cascade can be used to block access of RNA
polymerase to a gene, without damaging the target.
 Nuclease-deficient Cas9 can be exploited in a similar manner.

13. CRISPR-Cas9 Construct

14. crRNA
• Contains the guide RNA that locates the correct section of host DNA along with a
region that binds to tracrRNA (generally in a hairpin loop form) forming an active
complex.
tracrRNA
• Binds to crRNA and forms an active complex.
sgRNA
• Single guide RNAs are a combined RNA consisting of a tracrRNA and at least one
crRNA
Cas9
• Protein whose active form is able to modify DNA. Many variants exist with
differing functions (i.e. single strand nicking, double strand break, DNA binding)
due to Cas9's DNA site recognition function.
KEY COMPONENTS

15. 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.