Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM). However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT (Tethering EXonuclease T5 with FnCas12a)—a fusion strategy that significantly increased the knockout efficiency of FnCas12a in human cells at multiple genomic loci in three different cell lines. TEXT results in higher insertion and deletion efficiency than FnCas12a under different spacer lengths from 18 nt to 23 nt. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. Compared to other Cas12a orthologs, including AsCas12a and LbCas12a, TEXT achieves the highest on-targeting efficiency and shows minimal off-targeting effects at all tested sites. TEXT enhances the activity of FnCas12a nuclease and expands its targeting scope and efficiency in human cell genome engineering.

中文翻译：

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通过核酸外切酶融合改进 FnCas12a 基因组编辑

在目前报道的 Cas12a 直向同源物中，新弗朗西斯菌Cas12a (FnCas12a) 受原始间隔区相邻基序 (PAM) 的限制较少。然而，FnCas12a 核酸酶的活性在人类细胞中相对较低或检测不到，限制了其作为理想基因组工程工具的应用。在这里，我们描述TEXT（牛逼ethering EX onuclease ŧ5 与 FnCas12a)——一种融合策略，可显着提高 FnCas12a 在人类细胞中三个不同细胞系中多个基因组位点的敲除效率。在从 18 nt 到 23 nt 的不同间隔长度下，TEXT 比 FnCas12a 具有更高的插入和删除效率。深度测序显示 TEXT 显着增加了目标基因座的缺失频率和缺失大小。与其他 Cas12a 直向同源物（包括 AsCas12a 和 LbCas12a）相比，TEXT 实现了最高的靶向效率，并在所有测试位点显示出最小的脱靶效应。TEXT 增强了 FnCas12a 核酸酶的活性，扩大了其在人类细胞基因组工程中的靶向范围和效率。