5′ modifications to CRISPR–Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

Abstract

<jats:title>Abstract</jats:title> <jats:p>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.</jats:p>

Programmable downsizing of CRISPR-Cas9 activity for precise and safe genome editing

“…In our system, precise homozygous mutations can be obtained by repeated mono-allelic editing. Our finding is consistent with the context of previous studies suggesting that the increased on-target specificity of engineered Cas9s, truncated gRNAs, and gRNAs with a couple of guanine addition to the 5' end, is at least partially due to decrease in the activity of sgRNA-Cas9 complex 24,37,38 . FBS medium containing DMEM, 2 mM l-glutamine (Nacalai Tesque), 100 U/ml penicillin, 100 μg/ml streptomycin (P/S) (Nacalai Tesque) and 10% FBS.…”

“…Reducing amounts of the all-in-one plasmid or sgRNA-expressing plasmid failed to increase the clones with mono-allelic indels ( Fig describing that a few additional guanines at the 5' end may potentially interrupt the sgRNA-Cas9 activity 23,24 (Fig. 2b).…”

“…2). Next, addition of 15-base stretches of guanine [15G], cytosine [15C], adenine [15A], and thymidine [15T] to the 5' end of spacer sites was tested on the basis of previous reports describing that a few additional guanines at the 5' end may potentially interrupt the sgRNA-Cas9 activity 23,24 (Fig. 2b).…”

IFI16 knockdown in primary HIV-1 target cells

“…Off-targets of siRNA and shRNAs are mainly due to their instability. On the other hand, gRNAs are known to be much more stable due to their structure which includes R-loop dynamics ( Mullally et al., 2020 ).…”

Optimized guide RNA selection recommendations for usingspCas9 gene editing in human hematopoietic stem and progenitor cells

“…For RNP based gene editing, sgRNAs were initially in vitro transcribed (IVT). However, chemical synthesized sgRNA lacking the 5'triphosphate cap (15) outperform IVTsgRNAs by preventing RIG-1 mediated intracellular immune responses in primary cells thereby leading to fewer cellular off-target effects (21,22). Moreover, chemically synthesized RNAs contain modifications that improve RNA stability and have been shown to be more effective compared to IVTsgRNAs (15).…”