Figure 6.

A practical guideline for improving the HDR/NHEJ ratio using the CRISPR/Cas9 system. (A) A schematic representation of the relationship between the HDR and NHEJ activities and the threshold for the conformational checkpoint of Cas9. Changing the threshold for the conformational checkpoint often influences the HDR- and NHEJ-inducing activities of Cas9 to different extents. Therefore, there are ‘sweet spots’ where HDR and NHEJ can be upregulated and/or downregulated, respectively. (B) Alterations to improve the HDR/NHEJ ratio from the standard Cas9–gRNA complex. When a target genomic DNA sequence and a gRNA matches the target sequence, HypaCas9 should be used instead of WT Cas9 as the first option. If further improvements are required, eSpCas9(1.1) with and without the K810A or K855A mutation along with SpCas9-HF1 or SpCas9-HF4 can be useful in some cases as a second option. Alternatively, WT Cas9 combined with gRNAs of different lengths can also be tested. When a target sequence requires an extra 5′ guanine for a gRNA, WT Cas9 should be used as the first option, as the other Cas9 variants tend to have much lower activities in such situations. Some gRNAs with altered lengths combined with WT Cas9 can be used as a second option, but this can result in the simultaneous enhancement of NHEJ in many cases.