PRJNA562238
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 (&amp;Gamma;-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity–a &amp;Gamma;-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 &amp;Gamma;-Pcdh that cannot independently engagein homophilic trans-interactions, we find that &amp;Gamma;C4, 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 &amp;Gamma;C4 function that likely involves distinct molecular mechanisms.