Abstract
A key aim in exploiting CRISPR–Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9–gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3′ end. In contrast, we found that 5′ additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.
Highlights
The microbial CRISPR–Cas systems, and in particular type II-A Cas9, have found widespread utility as tools for sitespecific DNA and RNA recognition [1,2]
We repeated the magnetic tweezers (MT) assay (Figure 1C), to compare R-loop formation by St3Cas9 with SpCas9 using their corresponding crRNA:tracrRNA (Supplementary Figures S1 and S2, Tables S1 and S2). These are highly related Type II-A Cas9s (60% identity, 73% similarity) [29], which recognise similar protospacer adjacent motif (PAM) (Supplementary Figure S2A, Figure 2A) and can even exchange their crRNA:tracrRNA and retain activity [10], so we anticipated similar dynamics. crRNAs were synthesised using phosphoramadite chemistry and the 5 end was paired in the R-loop (Supplementary Figure S2A). tracrRNAs were synthesised by in vitro transcription (IVT) using T7 RNA polymerase, resulting in 3 unpaired 5 guanines (Supplementary Figures S1 and S2A)
GRNA engineering allows a wide range of enhancements to CRISPR–Cas activity [7]
Summary
The microbial CRISPR–Cas systems, and in particular type II-A Cas, have found widespread utility as tools for sitespecific DNA and RNA recognition [1,2]. They can be readily programmed by changing the spacer sequence of a crRNA that recognises a DNA target protospacer by forming an R-loop, binding to the target strand (TS) through Watson-Crick base pairing [3]. The single gRNA more commonly used in tool applications is a fusion of the tracrRNA and crRNA through a short RNA loop between the repeat-anti-repeat sequences in the Upper Stem (Figure 1) [4]. Our results indicate that SpCas R-loop formation and DNA cleavage activity is acutely sensitive to changes to the 5 end of the RNA, even when the addition is only two nucleotides
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