CRISPR-Cas systems provide bacteria with acquired immunity against viruses and plasmids. CRISPR loci contain repeating sequences separated by unique spacer sequences that are derived from invading genetic elements. Cas proteins process CRISPR RNA transcripts from the loci into small CRISPR RNAs that guide the degradation of invading nucleic acids based on sequence complementarity. This three-stage CRISPR-Cas immune response of adaptation, expression, and interference integrates new spacers and uses CRISPR RNAs to target matching foreign genetic elements. CRISPR-Cas systems are found in many bacteria and archaea and can be exploited for applications like bacterial typing, evolution studies, and generating phage resistance.

1. CRISPR- CAS System in
Phytopathogenic Bacteria – Acquired
Immunity
Presented by:
Sanjay Kumar

2. CRISPR- Cas system
 Term CRISPR was coined in 2002 by Jansen and coworkers to
reflect the particular structure of these loci
The CRISPR locus, first observed in Escherichia coli
90% of Archaea
40% of Bacteria
 CRISPR is an array of short repeated sequences separated by
spacers with unique sequences.
 The CRISPR can be found on both chromosomal and plasmid
DNA.
 The spacers are often derived from nucleic acid of viruses and
plasmids (an anti-virus system)
Clustered Regularly Interspaced Short Palindromic Repeats

3.  Repeat sequences are in the range of 21-48 bp, and
spacers are between 26-72 bp
 CRISPR loci is the presence of a conserved sequence,
called leader (20-534 bp), located upstream of the CRISPR
with respect to direction of transcription.
Cas proteins are the CRISPR-associated (cas) genes
(between 4-20) are almost always found in the vicinity of the
CRISPR region. These genes can be localized upstream or
downstream of the repeat/spacer region.
 Not all CRISPR loci have adjacent Cas genes and instead
rely on trans-encoded factors.

4. Structure of CRISPR

5. Role of CRISPR-Cas system in
bacterial adaptation
 The CRISPR-Cas defense system has the novel ability to
incorporate short sequences of non-self genetic material
known as spacers at specific locations within CRISPRs in the
host genome
 Spacers integrate primarily at one end (the leader end) of
the CRISPR locus (Bhaya D. et. al., 2011)
 The sequence on the viral genome that corresponds to a
spacer, is called a ‘protospacer’(23)
In several cases, there is a very short stretch of conserved
nucleotides in the immediate vicinity of the protospacer,
known as the Protospacer Adjacent Motif (PAM)

6.  PAM is a recognition motif required for
acquisition of the DNA fragment
 Spacers are transcribed and processed into small
non-coding RNAs
 Non-coding RNAs along with specific Cas protein
complexes can bind to incoming foreign genetic
material having a match of sequence
 This recognition process culminates in destruction
of the invading nucleic acid
 This surveillance and attack process makes use of
the previous exposure to a virus or plasmid to target
the incoming foreign DNA (or RNA)

7. Salmonella enterica (Shariat N. et. al. 2014, Appl. Environ. Microbiol. 80(2):430
Streptococcus thermophilus ( Deveau H et. al . 2010. Appl. Rev. Microbiol 64:475-93

8. CRISPR transcription
• CRISPR transcription initiates at the end of the locus
that contains the leader sequence, and the CRISPR
promoter might even reside within the leader itself
• The processing of CRISPR precursor RNA (pre-
crRNA) into small crRNAs is carried out by Cas proteins
• The source of the new spacer was first suggested to be
the mRNA. Presence of a gene coding for a putative
reverse transcriptase (RT) was reported in the vicinity of
the cas genes

9. • However, many CRISPR/Cas loci do not contain
such a gene. Thus, it is suggested that dsDNA is
the most likely source of new spacers
• The leader and other cas genes could participate
in the PAM recognition for acquisition of spacers
by base pairing and/or insertion through
recombination
• The Cas proteins are diverse genes present in the
vicinity of CRISPR. The core proteins, Cas1 to
Cas6, are characterized by their proximity to the
CRISPR.

10. Cas Proteins and their Functions

11. Cas proteins in Type I, II, and III CRISPR-Cas systems.

12. Mode of action of CRISPR-Cas
system -mediated resistance in
bacteria
CRISPR-mediated resistance has been proposed to involve
three stages:
i. CRISPR-adaptation or immunization or spacer
acquisition
ii. CRISPR-expression
iii. CRISPR-interference or immunity

13. i. CRISPR-adaptation/ immunization/ spacer acquisition:
• The invader DNA is encountered by the CRISPR-Cas
machinery and an invader-derived short DNA fragment
(Protospacer) is incorporated in the CRISPR array.
• It involves the recognition and subsequent integration of
spacers between two adjacent repeat units within the
CRISPR locus.
• Spacers appear to be integrated primarily at one end (the
leader end) of the CRISPR locus.
• It minimally requires two nucleases, Cas1 and Cas2, both
of which are universally present in genomes that have a
functional CRISPR-Cas system

14. ii. CRISPR-expression:
• A primary transcript or pre-CRISPR RNA (pre-
crRNA) is transcribed from the CRISPR locus by
RNA polymerase
• Then, specific endoribonucleases cleave the pre-
crRNAs into small CRISPR RNAs (crRNAs)
• Based on their function, these small RNAs are also
been referred to as prokaryotic silencing
(psiRNAs) or guide RNAs

15. iii. CRISPR-interference /immunity:
• The crRNAs form a multiprotein complex, called CASCADE
(CRISPR-associated complex for antiviral defense) in particular
organisms such as E. coli, can recognize and make specific base-
pairing with regions of incoming foreign DNA (or RNA) that have
perfect or almost perfect complementarity
• This initiates cleavage of the crRNA–foreign nucleic acid
complex, which inhibits the replication of the invading virus and
the host shows immunity to its attack
• If there are mismatches between the spacer and target DNA or if
there are mutations in the PAM, then cleavage is not initiated. In
this case, DNA is not targeted for attack, replication of the virus
proceeds, and the host is not immune to virus attack

16. CRISPR-mediated resistance in
Streptococcus thermophilus
Source: Bhaya, D. et.al. 2011, Annu. Rev. Genet. 45:273–97

17. Model of Type I, II, III and V CRISPR-Cas mechanism of action

19. CRISPR-Cas system in plant pathogen
Xanthomonas oryzae and evolution of
bacteriophages
Sensitivity of Xanthomonas oryzae strains Xo604 and Xo21 trains to phage
Xop411
• Both the strains carried a cassette that has a spacer exactly matching a
fragment of the phage (Xop411) genome
• But the strain Xo21 remains sensitive to phage Xop411 despite having a
matching spacer with the phage sequence
• Sequence analysis of CRISPR spacers of likely phage origin revealed that the
mutation in the motif adjacent to Xop411 proto-spacer, might be the reason of
phage’s ability to infect its host. Thus, Xop411 phage has evolved to evade the
bacterial resistance
• Similarly, Streptococcus thermophilus phages overcame CRISPR-based
resistance by single-nucleotide substitutions in their proto-spacers
(Semenova et al., 2009)

20. Applications of CRISPR-Cas system
 CRISPR has been used for bacterial strain typing
e.g. in Yersinia pestis, Corynebacterium diphtheriae, Streptococcus pyogenes,
Campylobacter jejuni, Streptococcus thermophilus and different species of
Lactobacillus
 The high variability of the CRISPR spacer content can be exploited for
phylogenetic studies
 CRISPR content analysis has become a routine procedure in bacterial genome
sequencing projects, as it is used in comparative analyses to study the evolution
of microbial populations and species (Bourgogne et. al., 2008)
 CRISPR interference can be used in the generation of phage resistant strains of
domesticated bacteria for dairy industry
 The CRISPR-Cas system can be exploited as a virus defense mechanism, to
reduce the dissemination of mobile genetic elements and reduce the
acquisition of undesirable traits such as antibiotic resistance genes and
virulence markers (Deveau et. al., 2008)

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