CRISPRs, the new antiviral

defense system in prokaryotes

Marcas O Muineachain, 106003290
Review Supervisor: Dr. Douwe van Sinderen
• Viruses and prokaryotes are ubiquitous on earth: dominant
predator – prey interaction in the biosphere
• Prokaryotes have developed defense mechanisms to fight
phage predation: Abortive infection; Adsorption inhibition;
DNA ejection inhibition; Restriction modification systems
• Recently discovered defense system titled Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPRs)
• CRISPR regions are a genetic locus where multiple functions
related to the bacterial response to phage infection are
located and organised
• CRISPRs found in almost all archaea and up to 50% of bacteria
• Between 1 and 18 CRISPR loci may be present in the genome,
depending on the organism e.g Streptococcus thermophilus
has 3 CRISPR loci
• Has been shown to confer acquired resistance against phage
infection in prokaryotes
 Structural components of the CRISPR system
• 4 Integral components of a CRISPR locus

• Repeat Sequences (Repeats)

• Spacer Sequences (Spacers)
• The leader Sequence (Leader)
• CRISPR Associated (CAS) genes
Repeats, spacers and the leader

Repeats: 24 – 47 bp; number of repeats per locus: 2 to 249; specific to a given CRISPR
locus and always identical in locus; highly conserved; short (5 – 7bp) palindrome in
larger groups (RNA secondary structure?); terminal repeat degenerate (GAAA) at
3’ end.
Spacers: flanked by 2 consecutive repeats; 26 – 72 bp; of constant and similar length;
always unique; not conserved; spacer sequences show homology to phage
sequences; bacteria acquire new spacers in response to phage predation; lead to
hypothesis that these spacers confer immunity to bacteria
Leader: 200 – 350 bp adjoining 5’ end of 1st repeat; A – T rich and non coding; no
open reading frame, not conserved; may act as a promoter
• CRISPR associated (Cas) genes
• always found adjacent to CRISPR loci
• exact number not known, varies between species
• 6 core Cas genes – Cas1 universal marker of CRISPR system

• Recent study identified Cas2 as an endoribonuclease

• Phylogenetic analysis of Cas genes – horizontal gene transfer
• Cas genes and the CRISPR loci together constitute a microbial immune
system
• Cas enzymatic machinery mediates phage resistance
• The mechanism of action of the CRISPR system :
Overview

• After phage attack, phage nucleic acids proliferate in the cell

• New virus particles created – lysis of most bacteria
• A small number of bacteria obtain phage-derived spacers (marked by
asterisk) leading to their survival due to CRISPR – mediated degradation of
phage nucleic acids
• A putative model for CRISPR action

• a spacer is acquired from the phage

• The repeat – spacer locus is transcribed into a long precursor RNA (repeats
assume a secondary structure)
• A complex of Cas proteins cleaves the CRISPR RNA precursor at each
repeat and maintains the cleavage products that contain the virus-derived
sequence
• A Cas protein, a helicase, assists mature CRISPR RNAs to serve as small
guide RNAs (sRNAs = spacer + 2 half repeats)

• sRNAs, when complexed with other Cas proteins, base pair with phage
nucleic acids, causing their degradation
• Similar to Eukaryotic RNA interference (RNAi) defense mechanism
• Phage response: mutation
• “arms race”
• Conclusion: Prokaryotes have evolved a nucleic acid
based immune system where the specificity is
determined by the CRISPR spacer sequence and the
resistance is mediated by the CAS enzymatic
machinery
• More research needed:
• Uncharcterised Cas genes
• How is the spacer acquired?
• What abut lysogeny?
• Do different CRISPR systems have different
functionalities?
• Now a popular study topic in scientific community:
more discoveries expected