Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
An innovative CRISPR-based editing system in primary human T cells is harnessed to engineer heterozygous variants across CARD11, a key regulator of immune signaling, in which heterozygous variants cause diverse IEI with variable penetrance.
Abstract
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)
Abstract Both genomic mutations and RNA editing contribute to functional complexity and drive adaptive evolution. Single-cell profiling offers deep insight into the cis-regulatory mechanisms underlying these variations. Using 13 025 single-cell Smart-Seq libraries from whole-body Drosophila melanogaster, we unexpectedly found that 94.0% of adenosine-to-inosine RNA editing sites and 92.8% of heterozygous single nucleotide polymorphismss (SNPs) with sufficient “unique fragment support” exhibit binary expression (0 or 1) in a single cell. The genotypes of representative heterozygous SNPs were validated by Sanger sequencing. Meanwhile, binary RNA editing itself is logically questionable due to elusive mechanism, compromised condition specificity, and untenable heterozygote advantage. This fact that for most cases in Smart-Seq, only a single allele (out of the various haplotypes) is finally maintained per cell, raises the following concern. Regardless of the biological or technical explanations like monoallelic transcriptional burst, dropout, or amplification bias that might account for this binary expression pattern, our findings conservatively indicate that Smart-Seq may not be good at analyzing molecular diversity and that the results need to be interpreted with caution.
Y. Duan, Jiyao Liu, Shiwen Xu et al.· Nucleic Acids Research· 0 citations
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.
Samantha Norris, Sai Goutham Reddy Yeddula, E. Su et al.· Frontiers in Genome Editing· 0 citations
Advances in high-throughput sequencing have associated millions of putative genetic variants with disease. However, scalable experimental methods to establish causal relationships between genetic variants and downstream transcriptional outcomes remain a major challenge. Single-cell methods that integrate genotyping with transcriptomic profiling provide a way to address this, but do not enable pre-sequencing enrichment of correctly edited cells, limiting scale. We present SELECT-seq (SNP Enrichment Leveraging Cas12a Targeting), a rapid method that allows SNP-specific PCR amplification and Cas12a-mediated fluorescence detection simultaneously with whole-transcriptome amplification. This one-pot workflow enables identification and enrichment of SNP-bearing single cells, making a rapid and scalable methodology for analysis of genotype-phenotype linkage avoiding laborious single cell cloning steps. As a proof of principle we show that SELECT-seq distinguishes U-2 OS and T-47D cell lines based on a PIK3CA (NM_006218.4:c.3463A>G) mutation while preserving transcriptome integrity. It physically enriches a rare NRF2 T80K (NM_006164.5:c.390C>A) mutant cells (6.7%) from a prime-edited pool, achieving 86% genotype accuracy, and shows 87.5% directional concordance in the transcriptomic effects compared with a clonal NRF2 T80K cell line. SELECT-seq thus provides a rapid, scalable and widely accessible approach for mapping genotype–phenotype relationships at single-cell resolution.
Sho Iwama, D. Gitterman, Timothy Brendler-Spaeth et al.· bioRxiv· 0 citations
It is shown that off-target editing and translocations vary widely between individual cells and organs, motivating the development of more sensitive and organ-specific safety assays for CRISPR therapies.
Alexandra Madsen, Niklas Selfjord, M. Martinez-Lage et al.· Nature Communications· 0 citations
To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies. Low editing efficiency of nonviral delivery in post mitotic tissues presents a challenge to the field of gene therapy. Here, authors dissect the genetic regulators of nonviral delivery in post mitotic retinal epithelial cells describe strategies for improved base editor delivery and editing.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· Nature Communications· 0 citations
A novel genome-wide CRISPR screening strategy that will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.
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