Biallelic editing of a lamprey genome using the CRISPR/Cas9 system.
Biallelic editing of a lamprey genome using the CRISPR/Cas9 system.
复制标题
使用 CRISPR/Cas9 系统对七鳃鳗基因组进行双等位基因编辑
DOI：
10.1038/srep23496
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发表时间：
2016-03-23
影响因子：
4.6
通讯作者：
Li W
中科院分区：
文献类型：
--
作者：
Zu Y;Zhang X;Ren J;Dong X;Zhu Z;Jia L;Zhang Q;Li W
七鳃鳗是无颌动物现存的代表。对七鳃鳗发育、生理和基因组的描述为脊椎动物性状的早期进化提供了重要的见解。然而，有效的手段，遗传操作在agnathan物种尚未开发，阻碍了功能的基因在这些重要的Evo-Devo模型的研究。在这里，我们报告了一个针对七鳃鳗基因组优化的CRISPR/Cas系统，并使用它来破坏东北七鳃鳗（Lethenteron morii）的基因组位点，效率在84~ 99%之间。通过对注射七鳃鳗幼体基因组DNA直接测序，在golden（gol）、kctd 10、wee 1、soxe 2和wnt 7 b等基因座上观察到的indel频率分别为68/69、47/56、38/39、36/37和36/42。这些indel经常出现在两个等位基因中。在golorkctd 10的CRISPR/Cas9处理中，38.6%或85.3%的靶向幼虫具有各自的隐性空样表型，进一步证实了两个基因座的破坏。针对Kctd 10的一个基本功能区设计的kctd 10 gRNA导致了空样表型和等位基因的框内突变。我们认为，基于CRISPR/Cas的方法具有在不太适合基于生殖系传播的方法的生物体中进行有效遗传扰动的潜力。
Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional studies of genes in these important Evo-Devo models. Here, we report a CRISPR/Cas system optimized for lamprey genomes and use it to disrupt genomic loci in the Northeast Chinese lamprey (Lethenteron morii) with efficiencies ranging between 84~99%. The frequencies of indels observed in the target loci ofgolden(gol),kctd10, wee1, soxe2, andwnt7b, estimated from direct sequencing of genomic DNA samples of injected lamprey larvae, were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively. These indels often occurred in both alleles. In the CRISPR/Cas9 treatment forgolorkctd10, 38.6% or 85.3% of the targeted larvae had the respective recessive null-like phenotypes, further confirming the disruption of both loci. Thekctd10gRNA, designed against an essential functional region of Kctd10, resulted in null-like phenotypes and in-frame mutations in alleles. We suggest that the CRISPR/Cas-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches.