This document provides an overview of the CRISPR-Cas immune system in bacteria and its applications. It discusses: 1. The history and components of the CRISPR-Cas system, including Cas proteins, CRISPR RNA, and protospacer adjacent motifs. 2. The three main types of CRISPR-Cas systems and their mechanisms of targeting DNA or RNA. 3. How engineered versions of Cas9 can be used for targeted genome editing and modulation of gene expression in mammalian cells. 4. Applications of CRISPR-Cas in microbiology such as genetic engineering of bacteria, gene repression/activation using deactivated Cas9, and developing sequence-specific antimicrobials.

2. CRISPR - cas : A New tool for Genetic
Manipulations from Bacterial Immunity
Systems
2
M. Raveendra Reddy
Ph D, UAS, GKVK, BANGLORE
Viral SS DNA
RNA Guide
CRISPR Cascade
subunit

3. Content…
1. Introduction
2. History
3. Components of CRISPR
4. Different Cas proteins and their function
5. Types of CRISPR cas system
6. Variants of Cas9
7. Targeted Genome editing in Mammalian
cells
8. Applications in Microbiology
9. Conclusion

4. CRISPR
• It represents a family of DNA repeats in most archaeal (~90%) and
bacterial (~40%) genomes provides acquired immunity against
viruses and phages.
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.

5. 5
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.

6. The spacer region of each CRISPR RNA base pairs with complementary
nucleic acids, driving cleavage or degradation by the Cas proteins (cas 1 and
Cas 2) within minutes of invasion.

7. 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.
In 2007 the first experimental evidence that CRISPR was an adaptive
immune system in Streptocous thermophilus was published (Hsu et al,
2014. Cell. 157 (6): 1262–78. )

8. 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)
Jennifer Doudna Emmanuelle Charpentier

9. Components of CRISPR
1. Protospacer adjacent motif (PAM)
2. CRISPR-RNA (crRNA)
3. trans-activating crRNA (tracrRNA)
4. Cas proteins

10. 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 10

11. 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. 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 .

13. 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.

14. 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.
Zetsche et al., (2015)
functioning of type II CRISPR
system

15. Includes CRISPR associated
complex for antiviral defense
(cascade) and cas3
CRISPR array is transcribed as
crRNA and processed to liberate
short mature crRNA
Cas6 endoribonuclease cleaves
the repeat sequence eight
nucleotides upstream of the spacer
sequence and liberates a small
crRNA containing a full spacer
flanked by partial repeats.
Type 1 CRISPR-Cas Systems

16. Efficient immunity requires
interaction between the first
eight nucleotides of the target
and the complementary
sequence of the crRNA guide at
the 5l end of the DNA:RNA
duplex.
A second requirement for
type I immunity is the presence
the protospacer-adjacent motif
(PAM).


17. For the type I-E system of E.
coli the PAM is an AWG
trinucleotide, and it is recognized
by CasA, a member of the
Cascade complex.
If both conditions are met, the
Cas3 ssDNA nuclease is
recruited by Cascade to cleave
the displaced DNA strand
within the target sequence and
degrade it with 3l →5l

18. -defined by presence of the RNA-
guided endonuclease Cas9
- the simplest of all CRISPR-Cas
systems
-type II CRISPR loci produce a
small trans-encoded crRNA
(tracrRNA) with a region of
complementarity to the repeat
sequence
gRNA = crRNA + tracrRNA
Type II CRISPR-Cas Systems

19. -The Cas9 nuclease binds the tracrRNA, which anneals to the
repeat sequences of the precursor crRNA.

20. Binding of Cas9 to the PAM favors unwinding of the target
sequence immediately upstream of the motif, allowing the crRNA
to probe for a matching sequence.
Productive annealing results
in formation of a
crRNA:target R-loop that
triggers cleavage by Cas9.
The enzyme has two
nuclease domains HNH and
RuvC - cuts one target DNA
strand.
The tracrRNA - cofactor of
Cas9 - DNA cleavage
Ruvc
HNH

21. 7
Type III CRISPR-Cas Systems
Type III CRISPR-Cas systems are possibly the most complex of all
Characterised by the presence of genes encoding Cas10 and repeat-
associated mysterious protein (RAMP) modules Csm for type III-A and
cmr for type III-B.
precursor processing is achieved by the Cas6 repeat-specific
endoribonuclease like in Type I System , but contrast to type I systems,
however, Cas6 is not part of the Cas10 complex

22. Two unique features of type III CRISPR-Cas systems are
 (a) that transcription across the target is required for immunity and
(b) both DNA and RNA targets are cleaved.
 In addition, only crRNA guides complementary to the coding DNA
strand—not those complementary to the template DNA strand—provide
effective immunity
In contrast to the other CRISPR-Cas, seed sequence has not identified.

25. Homology-directed repair (HDR) and nonhomologous end joining
(NHEJ) are DNA repair pathways exploited for targeted genome editing in
mammalian cells
Double-strand DNA breaks (DSBs) can increase recombination events in
the cleaved region up to 1,000-fold and programmable nucleases that can
cleave any sequence within the genome of interest are essential tools for
genome engineering.
Specific mutations can be introduced when cells are supplied with
exogenous DNA fragments as templates for recombination with the cleaved
site through HDR.
TARGETED GENOME EDITING IN MAMMALIAN CELLS

26. In the absence of a DNA template for HDR, DSBs are typically
repaired by the error-prone NHEJ pathway, in which insertion or
deletion mutations (indels) of various lengths are introduced into the
target locus.
These indels usually alter the gene’s open reading frame and therefore
lead to the generation of gene knockouts.
To reduce mutational effects of DSBs, biologists have looked for
programmable sequence-specific nucleases.
1. Meganucleases,
2. Zinc finger nucleases (ZFNs),
3. Transcription activator–like effector nucleases (TALENs).

27. Meganucleases:
Meganucleases are naturally occurring sequence-specific endonucleases found in
many microorganisms and plants.
They are characterized by a large DNA recognition site ranging from 12 to 40 base
pairs .
Meganucleases have not been widely adopted as genome editing tools…
…… because sequence specificity is hard to engineer, and despite the existence of
hundreds of meganucleases in nature, the probability of finding one able to cut a given
gene at a desired location is extremely slim.

28. Both ZFNs and TALENs are engineered nucleases in which a modular
DNA-binding domain is linked to the nuclease domain of the restriction
enzyme FokI.
Zinc Finger Nucleses
In ZFNs, the DNA-binding domain is a tandem array of different Cys2
His2 zinc fingers, each of which recognizes a three- base-pair sequence in
the target DNA.

29. In TALENs, the DNA-binding
domain is derived from DNA-binding
proteins found in Xanthomonas
bacteria and contains tandem array of
15-19 nucleotides.
Each module binds a single base
pair and consists of 34 amino acid
residues, with different amino acid
sequences recognizing different base
pairs
Trancription Activator Like Effector Nucleases (TALENS)

30. In both ZFNs and TALENs, sequence specificity is conferred by the
programmable DNA- binding domain.
In addition, the assembly of TALEN genes encoding large numbers of
TALE modules presents a challenge for molecular cloning as well as for
efficient viral delivery to target cells.
Difficulties in predicting and modulating ZFN and TALEN interaction
with the target DNA, as well as the laborious process of protein design,
limit the utility of these nucleases as tools for genome editing.

31. CRISPR/Cas 9 engineering tool
DNA cleavage is based on RNA/DNA pattern and not anymore on
Protein/DNA
Change require only in the first 20 nucleotides of the gRNA
(former crRNA)
Possibility of targeting multiple DNA sequences at once
Much more easier to target DNA sequence
13

32. Some limitations: off-target
Off-target: tolerance of Cas9 to mismatches in the RNA
guide sequence.
Limited by PAM motif
Depend of mismatch locations, lengths, compositions
Difficult to predict
14

33.  Mutations in the RuvC (D10A) and HNH (H840A) domains abolish
cleavage but do not impair binding of Cas9 to its targets.
 The catalytically dead version of Cas9 (dCas9) can be exploited for
multiple uses in molecular biology as a sequence-specific, RNA-
guided DNA-binding protein.
 Most important applications of dCas9 is modulation of transcription .
 Binding of dCas9 to promoter sequences or open reading frames can
prevent transcription initiation or elongation, respectively.
 Repression also can be enhanced.
EXPLOITING THE RNA-GUIDED DNA-BINDING ACTIVITY OF CAS9

35. 15
Variants of Cas9
Only one strand of DNA will be cut

36. Advantages of dCas9 modulation of gene expression (CRISPRi)
over RNAi technology
key distinctions between dCas9 and RNAi are that
(a) dCas9 downregulation of transcripts acts at the DNA level, in
contrast to posttranscriptional regulation by RNAi,
(b) dCas9 offers the possibility of upregulating gene expression,
allowing for discovery of gain-of-function phenotypes that are not
possible to obtain with RNAi, and
(c) dCas9 can act on miRNAs and any other genomic region.

37. Streptococcus pyogenes Cas9 :
-the standard cas9 used in researches
- PAM seq : 5’ NGG 3’
Staphylococcus aureus Cas9 :
Smaller than S. pyogenes Cas9
- PAM seq : 5’ NNGRRT 3’
Another important use of
dCas9 is visualization of specific
DNA loci in living cells by fusing it
with fluorescent proteins
Chen et al. fused dCas9 to
an enhanced green fluorescent
protein (eGFP) to interrogate the
spatiotemporal dynamics of DNA
sequences in living cells.

39. CAS9-BASED APPLICATIONS IN MICROBIOLOGY
1. Genetic Engineering
Bikard et al (2012) programmed the human pathogen
Streptococcus pneumoniae with CRISPR sequences that target capsule
genes (virulence factor).
CRISPR interference can prevent transformation of non
encapsulated, avirulent pneumococci into capsulated, virulent strains during
infection in mice.
Cell Host Microbe. 2012 Aug 16;12(2):177-86.

40. Result demonstrate that CRISPR interference can prevent the
emergence of virulence in vivo and that strong selective pressure for
virulence or antibiotic resistance can lead to CRISPR loss in bacterial
pathogens.
This approach has been successfully applied for genetic manipulation
in numerous bacterial species, including E. coli , Streptococcus
pneumoniae , Lactobacillus reuteri , Clostridium beijerinckii and
Streptomyces coelicolor as well as bacteriophages.

41. 2. dCas9-Based Gene Repression and Activation
Bikard et al. (2013) fused catalytically dead cas9 (dCas9) to
the ω subunit of RNA polymerase (RNAP) and expressed this fusion
(dCas9-ω) in an E. coli mutant lacking the gene for ω.
dCas9-ω occupies the place of ω in the RNAP complex, where it can
mediate the recruitment of RNAP to specific promoters using the
appropriate guide crRNA. This method enhances transcription.
Nucleic Acids Res. 41:7429–37

42. Sequence-Specific Antimicrobials
Cas9 chromosomal cleavage leads to toxicity to cell. This property is used for the
development of sequence specific antimicrobials by RNA guided nuclease.
In human microbiota, could selectively kill bacteria with a specific genetic
sequence. In contrast to conventional antibiotics, the Cas9 antimicrobial could be
programmed with a guide RNA matching a specific virulence gene sequence to kill
the bacterial pathogen harboring this gene but spare the rest of the microbiota.

43. Phagemids : (pDB114)
Phages can be used to deliver cas9-containing plasmids harboring
CRISPR RNA sequences that direct Cas9 to unique chromosomal loci to
achieve sequence-specific killing. (Staphylococcus aureus)
a) Only target cells with the cognate sequence are killed, whereas
nontarget cells survive the phagemid treatment.

44. Significant challenges must be overcome before Phagemid can be
developed as a suitable drug.
1. The most important obstacle is the method of delivery, given that
phagemids have the same narrow host. This substantially limits the
number of species that can receive the Cas9 plasmid; only those
expressing the appropriate phage receptor can be treated.
2. Phagemids are also immunogenic, and their large structure limits their
access to certain infection sites.
3. Furthermore, phagemids deliver Cas9 in the form of plasmid DNA,
which could be destroyed and neutralized by bacterial restriction
enzymes.

45. 45

46. Comparision between sgRNA and crRNA-tracrRNA
hetero duplex
Advantages
• Flexible targeting
• Sequence specific
• Efficient
• Precise cleavage
• Affordable
• Quick
• Multiplex guides
Limitation
 Cas9 is a large protein
 PAM – dependent design limitations
 Off –target cleavage

47. Cas9-based tools are a formidable asset for studies in basic
science, biotechnology, and medicine. Cas9-based technologies
are spreading at a remarkable speed that speaks to their simplicity
and efficiency. One outstanding objective is the precise
determination of off-target effects and their consequences.
 Although current data seem to indicate that these are not a
major obstacle, a better characterization of Cas9 cleavage is
required before the technology is used in humans.
Ethical considerations are a second outstanding issue
Conclusion