Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
Sci Rep. 2021 May 27;11(1):11163. doi: 10.1038/s41598-021-89546-0.
The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.
强大的基因组编辑工具酿脓链球菌 Cas9（SpCas9）需要三核苷酸 NGG 作为前导序列相邻基序（PAM）。由于 PAM 要求在狭窄的编辑窗口中发生，因此它是精确基因组编辑（如特定基因组位点的单个氨基酸替换和敲入）的限制。最近，开发了 SpCas9 变体（即 xCas9 3.7、SpCas9-NG 和 SpRY），它们可以识别人类细胞系中的 NG 二核苷酸或几乎任何其他 PAM 序列。在这项研究中，我们在盘基网柄菌中评估了这些变体。在针对 NG PAM 位点的靶向诱变的背景下，我们发现 SpCas9-NG 和 SpRY 比 xCas9 3.7 更有效。在 NA、NT、NG 和 NC PAM 位点的背景下，SpRY 在 NR（R=A 和 G）的编辑效率约为 60%，但在 NY（Y=T 和 C）的编辑效率小于 22%。我们成功地使用 SpRY 在特定基因座使用侧翼为 60 bp 同源臂的供体 DNA 产生了敲入。此外，我们实现了高达 97.7%的高效点突变。这项工作提供了工具，将大大扩展可用于敲除、敲入和精确点突变的基因座。
