Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants.
Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants.
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DOI：
10.1038/s41598-022-24810-5
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发表时间：
2022-11-27
影响因子：
4.6
通讯作者：
Onaka, Tatsushi
中科院分区：
文献类型：
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作者：
Yoshida, Masahide;Saito, Tomoko;Takayanagi, Yuki;Totsuka, Yoshikazu;Onaka, Tatsushi
用于在啮齿动物胚胎中产生敲入突变的CRISPR-Cas9方法产生了许多可能具有设计突变的FO代候选物。选择有希望的F0代的第一个任务是分析基因组DNA，其可能含有设计的和意外的突变的混合物。在我们的研究中，在产生Prlhr-Venus敲入报告基因小鼠的同时，我们发现除了F0代中设计的敲入突变之外，还发生了靶敲入等位基因附近的基因组重排、靶等位基因位点处的串联多拷贝和两种不同敲入等位基因的嵌合基因型。常规PCR和基因组测序不能检测嵌合现象，也不能区分设计的单拷贝敲入突变体和多拷贝插入突变体。然而，通过使用Southern印迹和下一代基于测序的RAISING方法的组合，在F0代中成功检测到这些突变体。在F1和F2代中，液滴数字PCR有助于建立菌株，尽管通过对F0代的分析将多拷贝错误地检测为一个拷贝。因此，这些方法的组合使我们能够选择有希望的F0代，并促进了设计菌株的建立。我们强调，仅关注基因敲入的阳性证据可能导致错误选择不良菌株。
The CRISPR-Cas9 method for generation of knock-in mutations in rodent embryos yields many F0 generation candidates that may have the designed mutations. The first task for selection of promising F0 generations is to analyze genomic DNA which likely contains a mixture of designed and unexpected mutations. In our study, while generating Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopies at a target allele locus, and mosaic genotypes for two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism nor discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In the F1 and F2 generations, droplet digital PCR assisted in establishing the strain, although a multicopy was falsely detected as one copy by analysis of the F0 generation. Thus, the combination of these methods allowed us to select promising F0 generations and facilitated establishment of the designed strain. We emphasize that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains.
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影响因子：
64.5
作者：
Yang H;Wang H;Shivalila CS;Cheng AW;Shi L;Jaenisch R
通讯作者：
Jaenisch R
影响因子：
12.3
作者：
Quadros RM;Miura H;Harms DW;Akatsuka H;Sato T;Aida T;Redder R;Richardson GP;Inagaki Y;Sakai D;Buckley SM;Seshacharyulu P;Batra SK;Behlke MA;Zeiner SA;Jacobi AM;Izu Y;Thoreson WB;Urness LD;Mansour SL;Ohtsuka M;Gurumurthy CB
通讯作者：
Gurumurthy CB
DOI：
10.15252/embj.201899243
发表时间：
2018-09-14
期刊：
The EMBO journal
影响因子：
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作者：
Lin YC;Pecetta S;Steichen JM;Kratochvil S;Melzi E;Arnold J;Dougan SK;Wu L;Kirsch KH;Nair U;Schief WR;Batista FD
通讯作者：
Batista FD
影响因子：
5.9
作者：
通讯作者：
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影响因子：
2.4
作者：
通讯作者：
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