Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.
Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.
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DOI：
10.1371/journal.pone.0116484
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Yokoyama WM
中科院分区：
文献类型：
--
作者：
Parikh BA;Beckman DL;Patel SJ;White JM;Yokoyama WM
细菌CRISPR-Cas9系统已被改装为基因组编辑工具。虽然最近的几份报告表明，可以成功地对小鼠进行基因组编辑，但对突变动物的详细表型和分子分析有限。在原核显微注射带有野生型Cas9或镍酶突变体(D10A)以及用于靶向酪氨酸酶(Tyr)基因的单个或成对引导RNA(SgRNA)的受精卵后，我们使用快速表型读数(眼睛和毛色)评估了小鼠的基因组编辑。在TYR中插入或缺失(INDELs)的突变小鼠可以有效地产生，而没有可检测到的脱靶切割事件。只有当供体DNA中的sgRNA识别位点被修饰时，才能通过同源重组(HR)对单个核苷酸进行基因校正。如果不对供体DNA进行修饰，则不会发生基因修复，因为CaS9的催化活性被完全抑制。我们的结果表明，等位基因嵌合体可以在小鼠中发生在Cas9介导的编辑之后，并且似乎与单细胞阶段的sgRNA切割效率相关。我们还表明，基于所采用的筛选策略，可能会忽略比预期更大的缺失。对我们实验中所有缺失的核苷酸进行的无偏分析表明，核苷酸缺失的最高频率聚集在预测的Cas9裂解位点周围，分布比预期的略宽。最后，对创始小鼠及其后代的额外分析表明，它们的总体健康、生育能力和基因变化的传播没有受到影响。这些结果为解释和预测CRISPR-Cas9介导的小鼠基因组编辑实验后的不同结果提供了基础。
The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.
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1.5
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