Gene Editing Research Center, Hebei University of Science and Technology, Shijiazhuang, PR China; Hebei University of Science and Technology, Shijiazhuang, PR China.
Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, USA; Hebei University of Science and Technology, Shijiazhuang, PR China.
CRISPR J. 2020 Dec;3(6):503-511. doi: 10.1089/crispr.2020.0073.
Among current reported Cas12a orthologs, 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 (ethering onuclease 5 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.
在目前报道的 Cas12a 同系物中，Cas12a（FnCas12a）受到的邻近基序（PAM）限制较小。然而，FnCas12a 核酸酶在人细胞中的活性相对较低或无法检测到，限制了其作为理想的基因组工程工具的应用。在这里，我们描述了 TEXT（与 FnCas12a 融合的醚化核酸酶 5）-一种融合策略，该策略可显著提高 FnCas12a 在三种不同细胞系中多个基因组位点的基因敲除效率。在从 18 nt 到 23 nt 的不同间隔长度下，TEXT 比 FnCas12a 具有更高的插入和缺失效率。深度测序表明，TEXT 可显著提高靶向位点的缺失频率和缺失大小。与其他 Cas12a 同系物，包括 AsCas12a 和 LbCas12a 相比，TEXT 在所有测试位点均实现了最高的靶向效率和最小的脱靶效应。TEXT 增强了 FnCas12a 核酸酶的活性，并扩展了其在人细胞基因组工程中的靶向范围和效率。
