Highly efficient base editing with expanded targeting scope using SpCas9‐NG in rabbits

Abstract

Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits.Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases. K E Y W O R D Sbase editor, CRISPR/Cas9, NG PAMs, rabbit | 589 LIU et aL.

Genetically Modified Rabbits for Cardiovascular Research

“…The standard protocol for generation of KO rabbits using CRISPR-Cas9 has been recently published (Yang et al, 2019). The techniques have been further refined (Liu et al, 2018(Liu et al, , 2020 so we can predict that in the next few years, more and more KO or KI rabbits will be made using this technology. Here we will briefly review some valuable KO rabbits created recently to discuss their usefulness in disclosing the molecular mechanisms of atherosclerosis.…”

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

“…We speculate that XMAS-TREE can be utilized in other applications not described in this study. For example, several groups have reported the generation of additional ABEs with non-NGG PAM specificities, narrower targeting windows, and reduced by-product formation [19,41,43]. Accordingly, future application of XMAS-TREE with these next-generation ABE variants will be straightforward.…”

Reduced off-target effect of NG-BE4max by using NG-HiFi system

“…7 In addition, a higher frequency of off-target editing events was also observed in Hoxc13 mutant rabbits generated by the NG-ABEmax system in our previous study. 6 In this study, significantly reduced off-target events and similar ontarget effects in Hoxc13 mutant rabbits were generated by NG-HiFi, which greatly improved the base editing specificity and expanded the genome targeting scope of base editing in human disease modeling and gene therapy in the future.…”

“…5 In addition, the NG-BE4max and NG-ABEmax systems are highly efficient tools for targeted base editing and have been used to precisely mimic human pathogenic mutations in rabbits. 6 However, a previous study showed that SpCas9-NG not only targeted the genome but also potentially increased off-target risk by generating new single-guide RNAs (sgRNAs). 7 In addition, a higher frequency of off-target editing events was also observed in Hoxc13 mutant rabbits generated by the NG-ABEmax system in our previous study.…”