Precise and broad scope genome editing based on high-specificity Cas9 nickases

Abstract

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by ‘tiptoeing’ over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects

“…These methods work with substantially less Cas9 dependent off-targets than nucleases, nevertheless, they also rely on cleavage, i.e., the nickase activity of SpCas9. The nickase versions of IFNs seem to exhibit the same sensitivity to the sequence contributions of the targets 32, 68 , thus applying target-matched IFN base and prime editors may decrease off-target editing of current editors to a non-detectable level and further. (vii) The identification of target-matched IFNs for a given target has proved to be straightforward here, still, a predictive algorithm, which could identify the target-matched IFNs for specific targets could further simplify this process and make it less labour intensive.…”

Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques

“…Besides, this approach has not yet been explored and used for the Cas9 protein structural diversity to identify good adaptive routes to higher fitness ( Gao et al, 2020 ). Concomitant with that, the need of multiple mutated residues in Cas9 has been predicted to improve specificity and efficiency by changing the conformational dynamics and biophysical properties of the iCas9 at different stages of its activity ( Palermo et al, 2017 , 2018 ; Boyle et al, 2021 ; Wang et al, 2021 ). Mostly, these residues mediate the allosteric communication of Cas9 (i.e., flexibility of the three conformational stages) and the RNA/DNA heteroduplex to ensure the proper positioning of the catalytic site and the proofreading step ( Yang et al, 2018 ).…”

Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing

“…69 It has been reported that the use of the inactivated RuvC domain of Cas-9 with sgRNA can reduce the off-target effect by 100 to 1500 times. 70 Moreover, the nuclease Cas-12a (previously known as Cpf1) is a type V CRISPR/Cas system that provides high genome editing efficiency. 71 Unlike the CRISPR/Cas-9 system, CRISPR/Cas-12a can process pre-crRNA into mature crRNA without tracrRNA, thereby reducing the size of plasmid constructs.…”