CRISPR/Cas9 interrogation of the murine Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (Γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity–a Γ-Pcdh hallmark–is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of mutants. Founders were crossed with C57BL/6J animals to generate G1 offspring, which were screened for heterozygous mutations using a custom amplicon approach to sequence target and potential off-target regions. Select mutants were made homozygous and analyzed by whole genome sequencing. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, as it is the only Γ-Pcdh that cannot independently engage in homophilic trans-interactions, we find that ΓC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved ΓC4 function that likely involves distinct molecular mechanisms.

哺乳动物Pcdhg基因簇编码由22种细胞黏附分子组成的蛋白家族——γ-原钙粘蛋白（gamma-Protocadherins, Γ-Pcdhs），该家族蛋白对神经元存活与神经环路形成至关重要。目前，作为Γ-Pcdhs标志性特征的亚型多样性，其在蛋白功能发挥中的必要性仍不明确。本研究采用CRISPR/Cas9系统降低亚型多样性，针对每个Pcdhg可变外显子使用混合单引导RNA（pooled single guide RNAs, sgRNAs）文库，以构建突变体的等位基因系列。将首建者（Founder）小鼠与C57BL/6J品系小鼠交配获得G1子代，通过定制扩增子测序法对靶区域及潜在脱靶区域进行测序，以此筛选杂合突变体。选取部分突变体构建纯合子品系，随后通过全基因组测序（whole genome sequencing, WGS）开展分析。对5个突变株系的分析结果表明，产后存活与神经元存活并不依赖亚型多样性。令人意外的是，作为唯一无法独立介导嗜同性反式相互作用的Γ-Pcdh，我们发现由Pcdhgc4编码的ΓC4是唯一的关键亚型。由于人类中仅其同源基因（orthologue）为唯一受到进化选择约束的PCDHG基因，本研究结果提示，ΓC4的功能具有进化保守性，且可能涉及独特的分子机制。