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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
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DOI：10.3389/fgeed.2026.1833024.png)
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Abstract
En 中文
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.
Keywords:
mouse model
CRISPR-Cas9
allele-specific targeting
Baker–Gordon syndrome
CRISPR-SWITCH
forced heterozygosity
monoallelic genome editing
synaptotagmin-1 (SYT1)
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