Generation of genetically modified mice using SpCas9-NG engineered nuclease
Generation of genetically modified mice using SpCas9-NG engineered nuclease
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DOI：
10.1038/s41598-019-49394-5
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发表时间：
2019-09-09
影响因子：
4.6
通讯作者：
Naito, Kunihiko
中科院分区：
文献类型：
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作者：
Fujii, Wataru;Ito, Haruka;Naito, Kunihiko
尽管通过在小鼠受精卵中使用CRISPR/Cas9介导的基因组编辑可以高效地产生遗传修饰的小鼠，但只有具有前间隔区邻近基序（PAM）序列的基因座是可靶向的。本研究调查了工程化化脓链球菌Cas9（SpCas 9-NG）在小鼠受精卵中的可用性。除了5 '-NGG序列之外，SpCas 9-NG将小鼠合子中的5'-NGA、5 '-NGC和5'-NGT序列识别为适于产生敲除小鼠的PAM。此外，SpCas 9-NG介导的基因组编辑能够产生小鼠受精卵中常规SpCas 9无法靶向的敲入小鼠。这些结果表明，受精卵中SpCas 9-NG介导的基因组编辑可用于在对应于NGN-PAM的基因座处产生敲除和敲入小鼠。
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.