Department of Entomology, University of Maryland, College Park, Maryland 20742.
Department of Entomology, University of Maryland, College Park, Maryland 20742
Genetics. 2020 Aug;215(4):1027-1037. doi: 10.1534/genetics.120.303269. Epub 2020 Jun 3.
In this manuscript, we report that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is highly efficient in the hemipteran The gene is well characterized in where mutation causes loss of eye pigmentation; is a reliable marker for transgenesis and other genetic manipulations. Accordingly, has been targeted in a number of nonmodel insects to establish tools for genetic studies. Here, we generated mutations in the () locus using CRISPR/Cas9. We found that is required for pigmentation throughout the body of , not just the ommatidia. High rates of somatic mosaicism were observed in the injected generation, reflecting biallelic mutations, and a high rate of germline mutation was evidenced by the large proportion of heterozygous G1s. However, mutations are homozygous lethal; G2 homozygotes lacked pigment dispersion throughout the body and did not hatch, precluding the establishment of a stable mutant line. Embryonic and parental RNA interference (RNAi) were subsequently performed to rule out off-target mutations producing the observed phenotype and to evaluate the efficacy of RNAi in ablating gene function compared to a loss-of-function mutation. RNAi knockdowns phenocopied homozygotes, with an unusual accumulation of orange granules observed in unhatched embryos. This is, to our knowledge, the first CRISPR/Cas9-targeted mutation generated in While we were unable to establish as a useful visible marker for , these findings are instructive for the selection of visible markers in nonmodel species and reveal an unusual role for an ortholog of a classic gene.
在本手稿中，我们报告了成簇规律间隔短回文重复（CRISPR）/Cas9 在半翅目昆虫中的高效性。该基因在 中得到了很好的表征，其中突变导致眼色素丧失；是转染和其他遗传操作的可靠标记。因此，已经在许多非模式昆虫中被靶向，以建立遗传研究工具。在这里，我们使用 CRISPR/Cas9 在 （）基因座中产生突变。我们发现 不仅是小眼，而是整个身体的色素形成所必需的。在注射的一代中观察到高比例的体细胞质突变，反映了双等位基因突变，并且通过大量杂合 G1s 证明了生殖系突变的高比率。然而， 突变是纯合致死的；G2 纯合子缺乏全身色素分散，并且没有孵化，排除了建立稳定突变系的可能性。随后进行了胚胎和母体 RNA 干扰（RNAi），以排除产生观察到的表型的脱靶突变，并评估 RNAi 与功能丧失突变相比在消除基因功能方面的功效。RNAi 敲低与 纯合子表型相同，未孵化的胚胎中观察到异常积累的橙色颗粒。据我们所知，这是首次在 中生成的 CRISPR/Cas9 靶向突变。虽然我们无法将 作为 的有用可见标记，但这些发现为非模式物种中可见标记的选择提供了启示，并揭示了经典 基因的同源物的不寻常作用。
