Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV 89557, USA.
Nevada State Public Health Laboratory, University of Nevada, Reno School of Medicine, Reno, NV 89557, USA.
J Cell Sci. 2020 May 11;133(9):jcs240705. doi: 10.1242/jcs.240705.
As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a ( AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequence-targeting CRISPR (cr)RNAs of 23 nt spacers are used, >70% of the founders obtained are edited. Moreover, the use of Mb3Cas12a tagged to monomeric streptavidin (mSA) in conjunction with biotinylated DNA donor template leads to high knock-in efficiency in two-cell mouse embryos, with 40% of founders obtained containing the desired knock-in sequences.
作为 Cas9 为基础的基因组编辑的替代和补充方法，Cas12a 由于其对 TTTV 原间隔基序 (PAM) 序列的严格要求，在哺乳动物细胞中尚未得到广泛应用。在这里，我们报告 Mb3Cas12a（AAX11_00205）可以基于 TTV PAM 序列有效地编辑小鼠基因组，其产生的大靶标缺失或插入数量最少。当使用 23 个核苷酸间隔的 TTTV PAM 序列靶向 CRISPR（cr）RNAs 时，超过 70%的获得的启动子是编辑的。此外，使用单体链霉亲和素 (mSA) 标记的 Mb3Cas12a 与生物素化 DNA 供体模板结合使用，可导致双细胞小鼠胚胎中的高敲入效率，40%的获得的启动子含有所需的敲入序列。
