Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity

Abstract

We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents.

Chimeric crRNAs Retaining Activity of Cas12a with Potential to Improve Specificity

“…When the mutation was located at the 5′‐end in PAM‐proximal seed region, both native crRNA and 8DNA were not able to cleave off‐target DNA (C1T), whereas they both could cleave the off‐target (C21A) containing the single mutation at the 3′‐end. This is consistent with the previous finding that the PAM‐proximal seed region is more sensitive to mutations than PAM‐distal region 26 . Interestingly, compared with native crRNA, 8DNA showed a lower activity on the off‐target (G10T) with the single mutation in the middle, indicating that the chimeric crRNA has higher target specificity than native crRNA (Figure 3(b)).…”

“…This result is reminiscent of the previous studies reporting that chimeric guide RNAs of Cas9 show decreased off‐target activity 26 . Destabilized target hybridization as well as altered binding kinetics by DNA replacement in crRNA could lead to improved target specificity 23–26 …”

“…Interestingly, compared with native crRNA, 8DNA showed a lower activity on the off‐target (G10T) with the single mutation in the middle, indicating that the chimeric crRNA has higher target specificity than native crRNA (Figure 3(b)). This result is reminiscent of the previous studies reporting that chimeric guide RNAs of Cas9 show decreased off‐target activity 26 . Destabilized target hybridization as well as altered binding kinetics by DNA replacement in crRNA could lead to improved target specificity 23–26 …”

“…We estimated the rate constant of the cleavage reaction of chimeric crRNAs by measuring time‐course increase of the cleavage product (Figure 1(d) and Supporting Information Figure S2). The peudo‐first‐order rate constant ( k obs ) of the cleavage reaction determined by following the previous study 26 was 0.51 min −1 when native crRNA was used (Figure 1(d)). The k obs values of the chimeric crRNAs containing 8 or less DNA residues (1DNA – 8DNA) were similarly determined as 0.51–0.81 min −1 .…”

“…Recently, the effect of DNA sugar backbone in guide RNAs of Cas9 has been investigated. Several groups including us have shown that a large number of RNA residues in the guide region of crRNA can be replaced with DNA without significantly compromising the Cas9 activity 23–26 . In addition, these chimeric crRNAs containing DNA residues commonly showed decreased off‐target activity, suggesting the benefit that they could bring into the field.…”