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.