优化近乎PAM-less腺嘌呤碱基编辑器提升编辑效率

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

目的探究替换近乎PAM-less腺嘌呤碱基编辑器(ABE)不同元件对其编辑效率的影响。方法在SpRY-ABEmax中,以Tad-8e替换ABEmax构建SpRY-ABE8e,在此基础上将Cas9蛋白变体SpRY与Tad-8e之间的连接肽3xGGS-XTEN-3xGGS改为长度缩短的PAPAPA连接肽。将ABEs和sgRNA表达质粒共转染人胚肾细胞HEK 293T,24 h后加入1.5μg/mL嘌呤霉素并于72 h后通过流式分选表达绿色荧光蛋白的细胞,提取细胞基因组并使用PCR扩增技术在预期产生编辑的位点进行扩增,采用Sanger测序并使用Beat工具预估各个位点编辑效率情况。结果与对照组SpRY-ABEmax相比,SpRY-ABE8e编辑效率提升,编辑窗口也扩大(P<0.05);通过使用长度缩短的连接肽可以在保持编辑效率提升的同时限制SpRY-ABE8e的编辑窗口。结论可通过替换PAM-less ABE不同元件来提升编辑效率,这将为今后基因治疗提供更多的工具选择。

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 1.5μg/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.