Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes
Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes
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DOI：
10.1242/dev.138057
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发表时间：
2016-08-01
期刊：
影响因子：
4.6
通讯作者：
Cho, Ken W. Y.
中科院分区：
文献类型：
--
作者：
Blitz, Ira L.;Fish, Margaret B.;Cho, Ken W. Y.
CRISPR/Cas9基因组编辑正在彻底改变遗传功能丧失分析，但在创建突变系时，技术限制仍然进展缓慢。首先，在传统的遗传育种方案中，携带突变等位基因的马赛克创始人动物通过异交产生F1杂合子。表型分析发生在F1杂交后的F2世代。因此，突变分析将需要多代人的研究。其次，当以必要基因为靶点时，创始人的高效突变往往是致命的，阻止了成熟动物的获得。降低突变水平可能会提高创始人的存活率，但会导致生殖系传播率更低、更具变异性。因此，研究致命突变的有效方法将是有用的。为了克服这些不足，我们引入了一种结合了有效的CRISPR突变和将突变的原始生殖细胞移植到野生型宿主中的方法--跳跃。用非洲爪哇进行了测试，我们发现，含有移植物的创始人可以高效地传递突变的等位基因。携带胚胎致死等位基因的F0动物杂交产生的F1后代概括了功能丧失的表型，绕过了整整一代的繁殖。我们预计，这种跨越将可以转移到其他物种。
CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce `leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.