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 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/ rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, given reports that it might not independently engage in 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.

14The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-15 Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which 16 isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a 17 CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled 18 sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels 19 at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that 20 postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, as it is the only γ-21Pcdh that cannot independently engage in homophilic trans-interactions, we find that γC4, encoded by 22Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained 23 in humans, our results indicate a conserved γC4 function that likely involves distinct molecular 24 mechanisms. 25 26 mammalian analogue to fly Dscam1, although their diversity is generated by differential isoform 38 expression via promoter choice rather than alternative splicing (Zipursky and Sanes, 2010). 39

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