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CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli.

Abstract：

:Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Beloglazova N,Kuznedelov K,Flick R,Datsenko KA,Brown G,Popovic A,Lemak S,Semenova E,Severinov K,Yakunin AF

doi

10.1093/nar/gku1285

subject

Has Abstract

pub_date

2015-01-01 00:00:00

pages

530-43

issue

1

eissn

0305-1048

issn

1362-4962

pii

gku1285

journal_volume

43

pub_type

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