利用 SpCas9-NG 工程化核酸酶生成基因修饰小鼠。
Generation of genetically modified mice using SpCas9-NG engineered nuclease.
机构信息
Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
出版信息
Sci Rep. 2019 Sep 9;9(1):12878. doi: 10.1038/s41598-019-49394-5.
Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5'-NGG sequence, SpCas9-NG recognized the 5'-NGA, 5'-NGC and 5'-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.
尽管通过使用 CRISPR/Cas9 介导的基因组编辑在小鼠受精卵中可以高效地产生基因编辑小鼠，但只有具有前导间隔相邻基序（PAM）序列的基因座才是可靶向的。本研究探讨了工程化酿脓链球菌 Cas9（SpCas9-NG）在小鼠受精卵中的可用性。除了 5'-NGG 序列外，SpCas9-NG 还识别小鼠受精卵中的 5'-NGA、5'-NGC 和 5'-NGT 序列作为 PAMs，适合用于生成基因敲除小鼠。此外，SpCas9-NG 介导的基因组编辑使得无法通过常规 SpCas9 靶向的小鼠受精卵中的基因敲入小鼠的生成成为可能。这些结果表明，SpCas9-NG 介导的基因组编辑在受精卵中可用于生成与 NGN-PAM 对应的基因座的基因敲除和基因敲入小鼠。
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