biostudies-literatureUnknown35(2)Weber JAClinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both <i>in vitro</i> and <i>in vivo</i>. We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency <i>in vivo</i>, Molecular therapy. Nucleic acids102172https://www.ebi.ac.uk/biostudies/studies/S-EPMC11229411biostudies-literatureTemporal restriction of Cas9 expression improves CRISPR-mediated deletion efficacy and fidelity.PMC11229411Carrell EMLang JFDavidson BLAlameh MGWeber JAfalseTemporal restriction of Cas9 expression improves CRISPR-mediated deletion efficacy and fidelity.Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both <i>in vitro</i> and <i>in vivo</i>. We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency <i>in vivo</i>, 2024-01-01T00:00:00Z2024 Jun2025-04-04T11:24:19.75Z2025-04-04T11:24:19.75ZS-EPMC112294113897869410.1016/j.omtn.2024.102172