biostudies-literatureUnknown32(4)Singh KComprehensive genome-wide studies are needed to assess the consequences of adeno-associated virus (AAV) vector-mediated gene editing. We evaluated CRISPR-Cas-mediated on-target and off-target effects and examined the integration of the AAV vectors employed to deliver the CRISPR-Cas components to neonatal mice livers. The guide RNA (gRNA) was specifically designed to target the factor IX gene (F9). On-target and off-target insertions/deletions were examined by whole-genome sequencing (WGS). Efficient F9-targeting (36.45% ± 18.29%) was apparent, whereas off-target events were rare or below the WGS detection limit since only one single putative insertion was detected out of 118 reads, based on >100 computationally predicted off-target sites. AAV integrations were identified by WGS and shearing extension primer tag selection ligation-mediated PCR (S-EPTS/LM-PCR) and occurred preferentially in CRISPR-Cas9-induced double-strand DNA breaks in the F9 locus. In contrast, AAV integrations outside F9 were not in proximity to any of ∼5,000 putative computationally predicted off-target sites (median distance of 70 kb). Moreover, without relying on such off-target prediction algorithms, analysis of DNA sequences close to AAV integrations outside the F9 locus revealed no homology to the F9-specific gRNA. This study supports the use of S-EPTS/LM-PCR for direct <i>in vivo</i> comprehensive, sensitive, and unbiased off-target analysis.Molecular therapy. Methods & clinical development101365https://www.ebi.ac.uk/biostudies/studies/S-EPMC11626537biostudies-literatureComprehensive analysis of off-target and on-target effects resulting from liver-directed CRISPR-Cas9-mediated gene targeting with AAV vectors.PMC11626537VandenDriessche TChuah MKSingh KFronza REvens HfalseComprehensive analysis of off-target and on-target effects resulting from liver-directed CRISPR-Cas9-mediated gene targeting with AAV vectors.Comprehensive genome-wide studies are needed to assess the consequences of adeno-associated virus (AAV) vector-mediated gene editing. We evaluated CRISPR-Cas-mediated on-target and off-target effects and examined the integration of the AAV vectors employed to deliver the CRISPR-Cas components to neonatal mice livers. The guide RNA (gRNA) was specifically designed to target the factor IX gene (F9). On-target and off-target insertions/deletions were examined by whole-genome sequencing (WGS). Efficient F9-targeting (36.45% ± 18.29%) was apparent, whereas off-target events were rare or below the WGS detection limit since only one single putative insertion was detected out of 118 reads, based on >100 computationally predicted off-target sites. AAV integrations were identified by WGS and shearing extension primer tag selection ligation-mediated PCR (S-EPTS/LM-PCR) and occurred preferentially in CRISPR-Cas9-induced double-strand DNA breaks in the F9 locus. In contrast, AAV integrations outside F9 were not in proximity to any of ∼5,000 putative computationally predicted off-target sites (median distance of 70 kb). Moreover, without relying on such off-target prediction algorithms, analysis of DNA sequences close to AAV integrations outside the F9 locus revealed no homology to the F9-specific gRNA. This study supports the use of S-EPTS/LM-PCR for direct <i>in vivo</i> comprehensive, sensitive, and unbiased off-target analysis.2024-01-01T00:00:00Z2024 Dec2025-04-03T23:55:46.489Z2025-04-03T23:55:46.489ZS-EPMC116265373965530910.1016/j.omtm.2024.101365