CRISPR technology

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CRISPR: A NEW DEFINITION FOR GENOME
EDITING
CRISPR: A NEW DEFINITION FOR GENOME
EDITING

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3. History
Yoshizumi Ishino in 1987, from Osaka University who accidentally
cloned part of a CRISPR together with the iap gene in E.coli
Francis Mojica (1995) studied CRISPR in the archaeal organism
Haloferax mediteranii and its function at the University of Alicante,
Spain.
The wide spread dispersal of CRISPR among prokaryotes was
presented at the Genomes 2000 meeting at the Institute Pasteur in
Paris by Jansen (named SPacer Interspersed Direct Repeat
(SPIDR)) and published by Mojica in the same year.
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4.  In 2007 the first experimental evidence that CRISPR was an
adaptive immune system in S. thermophilus was published (Hsu et
al, 2014. Cell. 157 (6): 1262–78. )
A 2010 study provided direct evidence that CRISPR-Cas cuts
both strands of phage and plasmid DNA in S. thermophilus
(Garneau J.E, 2010. Nature. 468 (7320): 67–71).
 Jennifer Doudna and Emmanuelle Charpentier (2012)
engineered Cas9 endonuclease into a more manageable two-
component system by fusing the two RNA molecules into a
"single- guide RNA" (Science. 337 (6096): 816–21).

5. CRISPR patent war: Biggest
Scientific Discovery
Prof. Jennifer doudna,
UC Berkeley, USA
Prof. Emmanuelle
Charpentier, Max plank
institute for infection
biology, Germany
Feng Zhang, Scientist, Broad
Institute, MIT, USA
(Berkeley News, 2018)

6. Diagram of the CRISPR prokaryotic antiviral defense mechanism

7. 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 plasmid.
•It represents a family of DNA repeats in most archaeal (90%) and
bacterial (40%) genomes provides acquired immunity against viruses and
phages.
Simplified diagram of a CRISPR locus.
The three major components of a CRISPR locus are shown: cas genes, a
leader sequence, and a repeat-spacer array. Repeats are shown as gray boxes
and spacers are colored bars. In addition, several CRISPRs with similar
sequences can be present in a single genome, only one of which is associated
with cas genes

9. Components of CRISPR
1. Protospacer adjacent motif (PAM)
2. CRISPR-RNA (crRNA)
3. trans-activating crRNA (tracrRNA)
4. Cas proteins
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10. The key steps of CRISPR-Cas immunity.
1)Adaptation: insertion of new spacers
into the CRISPR locus.
2)Expression: transcription of the
CRISPR locus and processing of
CRISPR RNA.
3)Interference: detection and
degradation of mobile genetic
elements by CRISPR RNA and Cas
protein(s).
2 MECHANISM

11. Cas genes
Small clusters of cas genes are often located next to CRISPR repeat-spacer
arrays. Collectively the 93 cas genes are grouped into 35 families based on
sequence similarity of the encoded proteins. 11 of the 35 families form
the cas core, which includes the protein families Cas1 through Cas9. A complete
CRISPR-Cas locus has at least one gene belonging to the cas core.
3
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 9

12. 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

13. CRISP
R
TYPES

14. Genome Editing
CRISPR/Cas9 systems are engineered versions of the Cas9 protein and guide
RNA. Typically, they are identical to the Streptococcus pyogenes type II CRISPR
systems, except that a single guide-RNA is used in place of the complementary
crRNAs and tracrRNAs of the natural CRISPR system, and the Cas9 protein is
codon-optimized for the cells intended to be transfected with the CRISPR/Cas9
system.
For example, CRISPR/Cas9 expression plasmids used to edit the genome of
human cells have codons optimized for human cells, and thus are called humanized
Cas9 (hCas9) .
CRISPR/CAS9

15. Component Function
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.
Repair template
DNA that guides the cellular repair process allowing
insertion of a specific DNA sequence
FUNCTIONS

16. •The Cas9 protein has six domains, REC I, REC II, Bridge Helix, PAM Interacting,
HNH and RuvC.
• The Rec I domain is the largest and is responsible for binding guide RNA. The role of
the REC II domain is not yet well understood.
• The arginine-rich bridge helix is crucial for initiating cleavage activity upon binding
of target DNA.
•The PAM-Interacting domain confers PAM specificity and is therefore responsible for
initiating binding to target DNA.
• The HNH and RuvC domains are nuclease domains that cut single-stranded DNA.
HNH = an endonuclease domain named for characteristic histidine and asparagine residues
RuvC = an endonuclease domain named for an E. coli protein involved in DNA repair

17. •The Cas9 protein remains inactive in the absence of guide RNA.
• In engineered CRISPR systems, guide RNA is comprised of a single strand
of RNA that forms a T-shape comprised of one tetraloop and two or three
stem loops.
•The guide RNA is engineered to have a 5 end that is complimentary to the′
target DNA sequence.

18. This artificial guide RNA binds to the Cas9 protein and, upon binding, induces a
conformational change in the protein. The conformational change converts the
inactive protein into its active form. The mechanism of the conformational
change is not completely understood. but hypothesis is that steric interactions or
weak binding between protein side chains and RNA bases may induce the change

19. •Once the Cas9 protein is activated, it stochastically searches for target DNA by binding
with sequences that match its protospacer adjacent motif (PAM) sequence.
•A PAM is a two- or three-base sequence located within one nucleotide downstream of the
region complementary to the guide RNA.
•When the Cas9 protein finds a potential target sequence with the appropriate PAM, the
protein will melt the bases immediately upstream of the PAM and pair them with the
complementary region on the guide RNA.
•If the complementary region and the target region pair properly,
the RuvC and HNH nuclease domains will cut the target DNA after the third nucleotide
base upstream of the PAM.

20. Natural CRISPR Pathway: 1. transcription of pre-crRNA and tracrRNA 2. binding of
tracrRNA to pre-crRNA 3. cleavage of guide RNA from pre-crRNA 4. binding of inactive
Cas9 nuclease to the guide RNA to produce the active Cas9 nuclease.
Engineered CRISPR: 1. transcription of Guide RNA as a single sequence 2. transcription and
translation of cas9 nuclease 3. binding of Guide RNA to Cas9 and Activation of Cas9

21. Mechanism of
CRISPR/Cas9 genome
editing
•CRISPR/Cas9 genome editing
requires a single guide (sg) RNA
that directs the Cas9
endonuclease to a specific region
of the genomic DNA, resulting in
a double strand break.
•By providing a donor DNA in
trans, a transgenic DNA can be
created, whereas in the absence of
a donor DNA, the double strand
break will be repaired by the host
cell, resulting in an insertion or
deletion, thus potentially
disrupting the open reading
frame of a gene.

22. Non-homologous end joining (NHEJ) is a pathway that repairs double-
strand breaks in DNA. NHEJ is referred to as "non-homologous" because the
break ends are directly ligated without the need for a homologous template, in
contrast to homology directed repair, which requires a homologous sequence to
guide repair.
Homology directed repair (HDR) is a mechanism in cells to repair double
strand DNA lesions. The most common form of HDR is homologous
recombination. The HDR mechanism can only be used by the cell when there is
a homologue piece of DNA present in the nucleus, mostly in G2 and S phase of
the cell cycle. Other examples of homology-directed repair include single-
strand annealing and breakage-induced replication.

24. •Deletions (using HDR with a template in which a deletion is engineered)
•Insertions (by providing a HDR template carrying a designer sequence)
•Knockouts (using NHEJ-mediated DSB repair)
•Transcriptional activation
•Transcriptional repression
•Fusion protein delivery (by direct or indirect recruiting of an effector
molecule of interest, through fusion, tethering, or by the use of guides
carrying protein-binding DNA sequences of interest).
•Imaging (using fluorophores)
•Epigenetic state alteration (using either epigenetic repressors such as the
LSD1 histone demethylase for interaction with distal enhancers, or
epigenetic activation using the p300 histone acetyltransferase)