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

Natalia Beloglazova,Ekaterina Semenova,Kirill A Datsenko,Konstantin Severinov,Konstantin Kuznedelov,Sofia Lemak,Greg Brown,Robert Flick,Ana Popovic,Alexander F Yakunin

doi:10.1093/nar/gku1285

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.

Highlights

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Clustered regularly interspaced short palindromic repeats (CRISPRs)-associated (Cas) proteins form an RNA-guided microbial immune system (CRISPR-Cas), which provides adaptive and inheritable immunity to the host against invading viruses and plasmids [1,2,3,4]
Recombinant E. coli Cascade complexes were purified from cells co-expressing Cas proteins with affinity tags either on Cse2 (CasB), Cas7 (CasC), Cas5 (CasD) or Cas6e (CasE) subunits and individual Cas proteins and a 61-nt-long guide RNA (crRNA) from a compatible plasmid harboring an engineered CRISPR array [7,13]
Bioinformatic analysis of potential secondary structures of crRNA5, 7, 11, 12 and 13 revealed that the inactive crRNA5 can form structures with the lowest predicted free energy compared to other crRNAs (Supplementary Figure S3) suggesting that these stable secondary structures can prevent specific binding of crRNA5 to Cascade. Be that as it may, our results indicate that the 5 handle of the E. coli crRNA is crucial for its binding to Cascade and subsequent DNA target recognition, whereas just five nucleotides of the 3 -handle are sufficient for crRNA function

Summary

Introduction

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins form an RNA-guided microbial immune system (CRISPR-Cas), which provides adaptive and inheritable immunity to the host against invading viruses and plasmids [1,2,3,4]. The CRISPR cassettes are transcribed and processed to generate short guide RNAs (crRNAs) containing individual spacers flanked with repeat fragments. The crRNAs form complexes with various Cas proteins and direct Cas nucleases toward target DNAs containing sequences matching crRNA spacers [5,6,7,8,9]. The most prevalent CRISPR type I system is further divided into six subtypes (I-A, I-B, I-C, I-D, I-E and I-F) depending on the presence of subtype-specific genes [2] Both type I and III systems involve the Cas family of endoribonucleases which cleave the repeat sequences of the crRNA precursor to generate short crRNAs with 8 nt of repeat sequence at their 5 ends (5 handle) [11,12].

Methods

Results

Conclusion