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Cas9 specifies functional viral targets during CRISPR–Cas adaptation
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Journal Article
Cas9 specifies functional viral targets during CRISPR–Cas adaptation
2015
Overview
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers).
Streptococcus pyogenes
Cas9 cleavage of the viral genome requires the presence of a 5′-NGG-3′ protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of
cas9
with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.
Bacterial CRISPR–Cas loci acquire short phage sequences called spacers that integrate between DNA repeats and how these viral sequences are chosen was unknown; in these studies of the type II CRISPR–Cas system of
Streptococcus pyogenes
, the Cas9 nuclease known to inactivate invading viral DNA was found to be required for the selection of functional spacers during CRISPR immunity.
Bacterial recognition of viral invaders
The once fanciful idea that bacteria might have immunological memory became accepted fact with the discovery that the CRISPR–Cas gene loci evolve rapidly to acquire short phage sequences, or spacers, which then integrate between CRISPR repeats and constitute a record of phage infection. These spacers are transcribed into small CRISPR RNAs (crRNAs) that are used to target the DNA of invading viruses. Two papers published in this issue of
Nature
describe molecular details about how bacteria create a DNA memory of the invading virus. Jennifer Doudna and colleagues show that the purified
Escherichia coli
Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA in a manner similar to retroviral integrases and DNA transposases. Cas1 is the catalytic subunit, while Cas2 increases integration activity; together they form the minimal machinery required for spacer acquisition. Luciano Marraffini and colleagues show that in the type II CRISPR–Cas system of
Streptococcus pyogenes
, the Cas9 nuclease that inactivates invading viral DNA using the crRNA as a guide is also required for the incorporation of new spacer sequences, by a yet to be determined mechanism.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ 45/23
/ 45/70
/ 45/77
/ Bacteria
/ Clustered Regularly Interspaced Short Palindromic Repeats
/ Clustered Regularly Interspaced Short Palindromic Repeats - genetics
/ Clustered Regularly Interspaced Short Palindromic Repeats - immunology
/ CRISPR-Associated Proteins - metabolism
/ CRISPR-Cas Systems - immunology
/ DNA
/ Enzymes
/ Genetics
/ Genomes
/ Humanities and Social Sciences
/ Microbiology and Parasitology
/ RNA
/ Science
/ Streptococcus pyogenes - enzymology
/ Streptococcus pyogenes - genetics
/ Streptococcus pyogenes - immunology
/ Streptococcus pyogenes - virology
/ Virology
