CRISPRs, the new antiviral defense system in prokaryotes

Marcas O Muineachain, 106003290Review 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 thermophilushas 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 acidbased immune system where the specificity isdetermined by the CRISPR spacer sequence and theresistance is mediated by the CAS enzymaticmachinery

• 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