Directed evolution of adenine base editors with increased activity and therapeutic application
Directed evolution of adenine base editors with increased activity and therapeutic application
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DOI：
10.1038/s41587-020-0491-6
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发表时间：
2020-04-13
影响因子：
46.9
通讯作者：
Ciaramella, Giuseppe
中科院分区：
文献类型：
--
作者：
Gaudelli, Nicole M.;Lam, Dieter K.;Ciaramella, Giuseppe
基础腺嘌呤碱基编辑器（例如，ABE7.10）使得能够实现可编程的A中心点T到G中心点C点突变，但在原代人类细胞中的挑战性基因座处编辑效率可能较低。在这里，我们使用腺苷脱氨酶变体文库进一步进化ABE7.10以创建ABE 8。在NGG原型间隔区邻近基序（PAM）位点，与ABE7.10相比，ABE 8在原型间隔区位置A5-A7处导致类似于1.5倍高的编辑，并且在位置A3-A4和A8-A10处导致类似于3.2倍高的编辑。非NGG PAM变体具有比ABE7.10高4.2倍的总体中靶编辑效率。在人类CD 34（+）细胞中，ABE 8可以在γ-珠蛋白基因HBG 1和HBG 2的启动子处重建天然等位基因，效率高达60%，导致胎儿血红蛋白的持续存在。在原代人T细胞中，ABE 8实现98-99%的靶向修饰，当在三个基因座上多重时，该靶向修饰得以维持。作为信使RNA递送，ABE 8在基因组DNA中不诱导显著水平的单向导RNA（sgRNA）非依赖性脱靶腺嘌呤脱氨，并且在细胞mRNA中诱导非常低水平的腺嘌呤脱氨。
The foundational adenine base editors (for example, ABE7.10) enable programmable A center dot T to G center dot C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in similar to 1.5x higher editing at protospacer positions A5-A7 and similar to 3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a similar to 4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34(+) cells, ABE8 can recreate a natural allele at the promoter of the gamma-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.