Engineering the mouse genome using Cas9-APOBEC base editing

Authors

• HK Lee
• MW Willi
• C Liu
• L Hennighuasen

Abstract

CRISPR-Cas9 genome editing is widely used, both in basic and translational research, and it holds promise in personalized medicine. However, since the current technology is based on Cas9 and its ability to introduce double stranded DNA breaks, it faces limitations. Introducing two independent allelic mutations will invariably lead to the deletion of the entire stretch of DNA between the two sites. This is the result of the double strand breaks introduced by Cas9 and the ensuing repair process. To bypass this impediment, we explored novel ‘base editing’ technologies, which rely on deaminases that convert cytidines to thymidine or adenosine to guanosine. we used two classes of base editors, BE3 with expanded PAM sequences and BE4 that converts cytidines to thymidine and ABE that converts adenosine to guanosine. To test the ability of these base editors to simultaneously mutate several allelic sites, we targeted three enhancers within a super-enhancer. 477 zygotes were injected with specific guide RNAs and the base editors and a total of 48 mice were born. Out of those, 31 carried the designed mutations. Most importantly, the conversion of adenosines to guanosines, using novel ABEs, was highly accurate and no additional and unexpected mutations were detected in the targeted area. This is the first study demonstrating that allelic mutations can be introduced into the genome using base editors. It is also the first study demonstrating that Adenosine Base Editors (ABEs) are highly accurate to introduce A to G transitions into the mammalian genome.

Scientific Focus Area: Genetics and Genomics

This page was last updated on Friday, March 26, 2021