Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline.
Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline.
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DOI：
10.1371/journal.pbio.3001478
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发表时间：
2021-12
期刊：
影响因子：
9.8
通讯作者：
Cooper KL
中科院分区：
文献类型：
--
作者：
Weitzel AJ;Grunwald HA;Weber C;Levina R;Gantz VM;Hedrick SM;Bier E;Cooper KL
高效的基因转换系统有潜力促进利用实验室小鼠研究复杂的遗传性状，如果作为“基因驱动”实施，可以限制野生啮齿动物种群造成的生物多样性丧失和疾病传播。我们之前表明，在序列靶向 CRISPR/Cas9 双链 DNA 断裂 (DSB) 后，这种从杂合到纯合的基因转换系统在雌性小鼠种系中是可行的。然而，在雄性种系中，所有 DSB 均通过末端连接 (EJ) 机制进行修复，形成“插入/删除”(indel) 突变。这些观察结果表明，将 Cas9 表达的时间安排与减数分裂 I 一致对于同源染色体对齐和染色体间同源定向修复 (HDR) 机制占主导地位的条件至关重要。在这里，我们使用 Spo11 基因座的 Cas9 敲入等位基因，表明 Cas9 的减数分裂表达确实介导雄性和雌性种系中的基因转换。然而，雄性和雌性种系中 HDR 和 indel 突变的频率较低，表明 Spo11 基因座的 Cas9 表达水平可能太低，无法有效形成 DSB。我们认为，在早期减数分裂 I 期间启动更强大的 Cas9 表达可能会提高基因转换的效率，并进一步提高雄性和雌性小鼠的“超孟德尔”遗传率。这项研究表明，虽然在雄性和雌性小鼠中，减数分裂 I 期间 Cas9 的表达促进基因型转换（CRISPR“基因驱动”的潜在机制），但 Cas9 蛋白的时机和高水平对于实现稳健的效率至关重要。
Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice. This study shows that while Cas9 expression during meiosis I promotes genotype conversion - the mechanism underlying CRISPR ’gene drive’ - in both male and female mice, timing and high levels of Cas9 protein are critical to achieve robust efficiency.
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DOI：
10.1042/bj20080413
发表时间：
2009-02-01
期刊：
The Biochemical journal
影响因子：
--
作者：
Mahaney BL;Meek K;Lees-Miller SP
通讯作者：
Lees-Miller SP
DOI：
10.1126/science.aaa5945
发表时间：
2015-04-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gantz VM;Bier E
通讯作者：
Bier E
影响因子：
4.5
作者：
Enguita-Marruedo, Andrea;Martin-Ruiz, Marta;Page, Jesus
通讯作者：
Page, Jesus
DOI：
10.1016/j.mrfmmm.2010.02.004
发表时间：
2010-06-01
影响因子：
2.3
作者：
Ahmed, Emad A.;Philippens, Marielle E. P.;de Boer, Peter
通讯作者：
de Boer, Peter
影响因子：
14.8
作者：
Grunwald, Hannah A.;Weitzel, Alexander J.;Cooper, Kimberly L.
通讯作者：
Cooper, Kimberly L.