Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease
Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease
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DOI：
10.1007/s11033-022-08054-0
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发表时间：
2022-11
影响因子：
2.8
通讯作者：
Teppei Goto;Kyoko Yogo;S. Hochi;M. Hirabayashi
中科院分区：
文献类型：
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作者：
Teppei Goto;Kyoko Yogo;S. Hochi;M. Hirabayashi
背景Cas9核酸酶以Cas9蛋白或mRNA的形式与CRISPR引导RNA(gRNA：双crRNA：trrRNA或嵌合的单引导RNA)一起传递，或以同时编码Cas9和CRISPR gRNA的质粒包的形式传递。方法和结果我们直接比较了通过原核注射Cas9以蛋白质、mRNA或质粒DNA的形式产生Foxn1基因座纯合突变的大鼠囊胚的效率。为了高效地生产带有纯合子Foxn1突变的大鼠囊胚，原核注射CaS9蛋白60 ng/µL可能是最佳的。虽然mRNA组的囊胚收获率高于蛋白质组和质粒DNA组，但基因型分析表明，蛋白质组、mRNA组和质粒DNA组中分别有63.6%、8.7-20.0%和25.0%的囊胚是纯合子突变。在60 ng/µL蛋白组中产生纯合突变囊胚的高效率可能与第一次卵裂前启动的初级基因组编辑有关。在大多数情况下，靶Foxn1基因座的纯合子突变是通过非同源末端连接或微同源介导的末端连接的缺失和修复来触发的。结论蛋白形式的Cas9核酸酶与CRISPR gRNA(核糖核蛋白)共注射入大鼠受精卵原核后，可迅速进入靶基因组位置并诱导双链断裂，从而获得高效率的纯合突变株。
BackgroundThe Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA.Methods and resultsWe directly compared the efficiency of producing rat blastocysts with homozygous mutations of theFoxn1locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygousFoxn1mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7–20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the targetFoxn1locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction.ConclusionsThe Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.