Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.
Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.
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DOI：
10.1186/s13059-017-1220-4
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发表时间：
2017-05-17
期刊：
影响因子：
12.3
通讯作者：
Gurumurthy CB
中科院分区：
文献类型：
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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
条件性基因敲除小鼠以及表达重组酶、报告基因和诱导型转录激活因子的转基因小鼠对许多遗传学研究至关重要，并且占所创建小鼠模型的90%以上。条件性基因敲除小鼠是利用在胚胎干细胞中进行同源重组的劳动密集型方法产生的，并且仅适用于约25%的所有小鼠基因。通过随机基因组插入方法产生的转基因小鼠存在表达不可靠的问题，因此需要靶向插入模型。尽管据报道基于CRISPR的策略可通过将靶向组分直接一步递送至受精卵来创建条件性和靶向插入等位基因，但这些策略效率相当低。
在此我们描述了Easi - CRISPR（带有单链DNA插入片段的高效添加 - CRISPR），这是一种靶向策略，其中长单链DNA供体与预组装的crRNA + tracrRNA + Cas9核糖核蛋白（ctRNP）复合物一起被注射到小鼠受精卵中。我们针对十几个基因座表明，Easi - CRISPR在8.5% - 100%的所得存活后代中产生正确靶向的条件性和插入性等位基因。
Easi - CRISPR解决了动物基因组工程的主要问题，即靶向DNA盒插入的低效性。该方法很稳健，对所有测试的基因座都成功。它具有通用性，可产生条件性和靶向插入等位基因。最后，它效率很高，因为平均仅处理50个受精卵就足以在多达100%的存活后代中产生正确靶向的等位基因。因此，Easi - CRISPR为构建大规模Cre - LoxP动物资源提供了一种综合手段。
本文的在线版本（doi:10.1186/s13059 - 017 - 1220 - 4）包含补充材料，授权用户可获取。
Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring. Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources. The online version of this article (doi:10.1186/s13059-017-1220-4) contains supplementary material, which is available to authorized users.