CRISPR-SWITCH (silent mutations with intention to create heterozygotes): a strategy for monoallelic genome editing and generation of a Syt1-D365E mouse model of Baker-Gordon syndrome.

Abstract

Precise control of allelic outcomes remains a major limitation of CRISPR-Cas9 genome editing, particularly for genes in which biallelic modification is lethal or confounds disease modeling. Here, we present CRISPR-SWITCH (Silent mutations With Intention To Create Heterozygotes), a genome engineering strategy that enables deliberate monoallelic editing by exploiting allele-specific CRISPR targeting. CRISPR-SWITCH operates through the initial introduction of a synonymous nucleotide substitution that creates a unique guide RNA recognition site, allowing subsequent selective editing of the engineered allele while preserving the wildtype copy. We applied CRISPR-SWITCH to generate a mouse model of Baker-Gordon syndrome, a dominant-negative neurodevelopmental disorder caused by pathogenic variants in synaptotagmin-1 (SYT1). Conventional CRISPR-Cas9 editing of the Syt1 locus produced complex allelic outcomes characterized by biallelic editing and mosaicism, preventing reliable generation of the defined heterozygous genotype required for disease modeling. In contrast, CRISPR-SWITCH enforced heterozygosity by first introducing a synonymous Y364Y mutation and then selectively targeting this allele to install the pathogenic D365E variant. This approach produced viable Syt1-D365E mice with exclusive monoallelic genome editing, predictable preservation of a wildtype allele, and balanced (1:1) expression of mutant and wildtype transcripts. Together, these results demonstrate proof-of-principle that CRISPR-SWITCH can enforce heterozygosity at endogenous loci and enable the generation of viable mammalian models for dominant-negative and dosage-sensitive genetic disorders.