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

Jan 10, 2020FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Highly efficient gene editing with a broader targeting range using SpCas9-NG in rabbits

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Abstract

NG-BE4max and NG-ABEmax systems achieve targeted mutation efficiencies of 75%-100% and 80%-100% in rabbit embryos.

Base editors composed of engineered SpCas9 variants enhance the ability to edit specific DNA sequences.
The newly developed SpCas9-NG variant recognizes minimal NG protospacer adjacent motifs, expanding the range of target sites.
NG-BE4max demonstrated high efficiency in converting cytidine to thymidine, while NG-ABEmax effectively converted adenine to guanine.
Both base editor systems successfully created new rabbit models with precise point mutations.
NG-ABEmax was able to replicate a specific human mutation in rabbits, which was challenging for traditional base editors.

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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.

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

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