Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation
Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation
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DOI：
10.1186/s13059-019-1776-2
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发表时间：
2019-08-26
期刊：
影响因子：
12.3
通讯作者：
Burgio, Gaetan
中科院分区：
文献类型：
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作者：
Gurumurthy, Channabasavaiah B.;O'Brien, Aidan R.;Burgio, Gaetan
CRISPR-Cas9基因编辑技术促进了基因敲除小鼠的产生，为繁琐耗时的传统胚胎干细胞方法提供了替代方案。早期的研究报道了通过显微注射2个单向导RNA（sgRNA）和2个单链寡核苷酸作为供体（本文称为“双供体floxing”方法）产生条件性敲除（cKO或floxed）等位基因的效率高达16%。结果我们重新评估了来自世界各地20个实验室的联盟的双供体方法。该数据集由56个遗传基因座、17，887个受精卵和1718只活产小鼠组成，其中仅15只（0.87%）小鼠含有cKO等位基因。我们对数据集进行了统计分析和机器学习算法，结果表明，所分析的因素都不能预测这种方法的成功。我们测试了一些较新的方法，这些方法在18个基因座上使用单供体DNA，而双供体方法未能产生cKO等位基因。我们发现，单供体方法比双供体方法效率高10至20倍。结论我们提出双供体方法缺乏效率，因为它依赖于顺式的两个同时重组事件，这一结果与普遍伴随的不期望的编辑事件相比相形见绌。使用单供体DNA的方法是相当有效的，因为它们仅依赖于一个重组事件，并且在没有意外突变事件的情况下正确插入供体盒的概率要高得多。因此，单供体方法为cKO动物模型的常规生成提供了更高的效率。
Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.