Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

Regina Shebanova,Konstantin Severinov,Ruslan Vasilev,Nikita Shebanov,Ivan Tarassov,Vadim Shmanai,Konstantin Kuznedelov,Ilya Mazunin,Egor Ulashchik,Nina Entelis,Natalia Nikitchina,Vladimir Mekler,Olga Sharko

doi:10.1093/nar/gkab1227

Abstract

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

Highlights

The CRISPR-Cas systems provide adaptive immunity to bacteria and archaea [1,2,3]
Though split CRISPR RNAs (crRNAs) are functional at higher concentrations than fullsized crRNAs, we propose that the use of split crRNAs for specific recognition and cleavage of DNA templates opens ways for future mechanistic research and practical applications of Cas12a effectors that require multiplexing and/or simultaneous interrogation of multiple targets
Recombinant AsCas12a was tested with a set of chemically synthesized crRNAs truncated from the 5 -end, i.e., harbouring a scaffold moiety of different lengths and a constant 20-nt spacer moiety in an in vitro DNA cleavage assay

Summary

Introduction

The CRISPR-Cas systems provide adaptive immunity to bacteria and archaea [1,2,3]. They consist of CRISPR (clustered regularly interspaced short palindromic repeats) arrays and CRISPR-associated (Cas) proteins (Figure 1A). CRISPR arrays are transcribed into pre-CRISPR RNAs, which are processed at the repeat sequences generating short CRISPR RNAs (crRNAs). To prevent self immunity that could arise through recognition of CRISPR array spacers from which crRNAs are derived, many effectors require, in addition to crRNA spacer-target protospacer complementarity, the presence of a short Protospacer Adjacent Motif (PAM) that is recognized by the effector protein. Since there is no PAM sequence in the repeat, targeting of the array is prevented

Methods

Results

Conclusion