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
CRISPR-SWITCH is presented, a genome engineering strategy that enables deliberate monoallelic editing by exploiting allele-specific CRISPR targeting and can enforce heterozygosity at endogenous loci and enable the generation of viable mammalian models for dominant-negative and dosage-sensitive genetic disorders.
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.
Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor’s genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.
D. Skoczek, Jerzy Hohendorff, Maciej T. Małecki et al.· Human Genetics· 0 citations
Heterozygous single-nucleotide variants cause many inborn errors of immunity (IEI), yet most functional genomics tools generate homozygous edits, limiting insight into dominant-negative (DN) and dosage-dependent variant effects. Expression imbalance of the mutant and wild-type alleles can further complicate genotype—phenotype relationships, and has been nearly impossible to interrogate experimentally. To overcome this, we built a scalable platform to engineer heterozygous variants in primary immune cells and directly link allelic expression bias to phenotype.
We harnessed an innovative CRISPR-based editing system, helicase-assisted continuous editing (HACE), in primary human T cells to engineer heterozygous variants across CARD11, a key regulator of immune signaling, in which heterozygous variants cause diverse IEI with variable penetrance. In parallel, we developed single-cell Allele-Integrated Multi-omics sequencing (sc-AIMseq), which integrates transcriptomics, surface proteomics, and quantification of mutant and wild-type allele expression at single cell resolution.
HACE screens recovered known pathogenic DN variants in CARD11 and revealed novel DN and haploinsufficient variants not evident in homozygous models. sc-AIMseq of HACE-edited T cells uncovered marked cell-to-cell variability in CARD11 allelic expression bias, which dramatically influenced T cell phenotype. We also performed sc-AIMseq on PBMCs from multiple patients with DN CARD11 mutations, which revealed that CARD11 allelic expression bias is cell-type dependent, providing a mechanistic basis for variable penetrance.
This work establishes a scalable strategy to model heterozygous genetic disorders in primary and patient-derived immune cells, and dissect their variable penetrance. By directly coupling heterozygous variant engineering, allelic expression bias, and phenotype at single-cell resolution, our approach enables unprecedented functional variant interpretation and advances precision immunology.
Z.H.W. has received research funding from NCI F30CA298572 and the Melanoma Research Foundation.
Technological Innovations in Immunology (TECH)
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