Optimize nearly PAM-less adenine base editor to improve editing efficiency

Abstract

Objective To explore the effects of replacing different components of PAM-less adenine base editor (ABE) on its editing efficiency. Methods ABEmax was replaced with Tad-8e in SpRY-ABEmax to construct SpRY-ABE8e. Then the connecting peptide 3xGGS-XTEN-3xGGS between Cas9 protein variant SpRY and Tad-8e was shortened to PAPAPA. ABEs and sgRNA expression plasmids were co-transfected into HEK 293T cells. After 24 hours, cells were incubated with 1.5 μg/mL puromycin. After 72 hours, the cells expressing green fluorescent protein were flow sorted and the cell genome was extracted and the expected editing sites were amplified by PCR technology. Sanger sequencing was performed and Beat tool was used to estimate the editing efficiency of each site. Results Compared with control group, SpRY-ABEmax, SpRY-ABE8e has higher editing efficiency and expanded editing window(P＜0.05); editing window of SpRY-ABE8e can be limited to a certain extent while maintaining editing efficiency by using the shortened linker peptide. Conclusions The editing efficiency can be improved by replacing different components of PAM-less ABE, which will provide more candidate tools for gene therapy in the future.

Keywords

• adenine base editor|spry|abe8e|editing efficiency