Turner Ashley N, Andersen Reagan S, Bookout Ivy E, Brashear Lauren N, Davis James C, Gahan David M, Davis James C, Gotham John P, Hijaz Baraa A, Kaushik Ashish S, Mcgill Jordan B, Miller Victoria L, Moseley Zachariah P, Nowell Cerissa L, Patel Riddhi K, Rodgers Mia C, Patel Riddhi K, Shihab Yazen A, Walker Austin P, Glover Sarah R, Foster Samantha D, Challa Anil K
Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
J Genet. 2018 Dec;97(5):1315-1325.
Nodal-related protein (ndr2) is amember of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the gene in the zebrafish () lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish and observed cyclopia in the injected (G) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an nuclease assay and analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the gene.
节点相关蛋白（ndr2）是转化生长因子β超家族因子的成员，是斑马鱼胚胎中枢神经系统腹侧中线模式形成所必需的。在人类中，编码节点的基因突变会导致全前脑畸形和内脏异位。斑马鱼中该基因的突变会导致类似的表型，包括内侧底板缺失、腹侧前脑发育严重缺陷和独眼畸形。该基因的等位基因在研究中枢神经系统腹侧结构的模式形成中很有用。斑马鱼中已报道了15种不同的等位基因，其中8种是通过化学诱变产生的，4种是辐射诱导的，其余等位基因是通过随机插入、基因靶向（TALEN）或未知方法获得的。因此，大多数突变位点是随机的，无法事先预测。我们使用化脓性链球菌的CRISPR-Cas9系统，靶向斑马鱼所有三个外显子中的不同区域，并在注射的（G）胚胎中观察到独眼畸形。我们表明，使用sgRNA-Cas9核糖核蛋白（RNP）复合物可在注射的（G）胚胎中引起明显的独眼畸形表型。使用桑格测序的靶向聚合酶链反应扩增子分析表明，大多数等位基因具有小的插入缺失，导致移码。序列信息与ndr2活性的丧失相关。在本研究中，我们使用核酸酶测定法验证了多个CRISPR靶点，并使用胚胎进行了分析。我们描述了一个导致保守的末端半胱氨酸编码序列缺失的特定突变等位基因。这项研究再次证明了CRISPR-Cas9系统在产生结构域特异性突变方面的实用性，并为该基因的结构功能提供了进一步的见解。
