biostudies-literatureUnknownSabol ALNIDDK NIH HHSNIEHS NIH HHSNCI NIH HHSNIGMS NIH HHSEngineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel <i>Streptococcus pyogenes</i> Cas9 variants with both increased and decreased DNA bibioRxiv : the preprint server for biology2025.12.11.693673https://www.ebi.ac.uk/biostudies/studies/S-EPMC12710652biostudies-literatureAnti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.PMC12710652R35 GM136354R01 DK132900R01 GM132825R01 GM137606P30 CA014051P30 ES002109Choudhary ABarybin AMButty VLHarris RMChaudhary SXu LQShoulders MDLocicero CMSeverance ZCLee CMengiste AASreekanth VHendel SJKailass KSingh PSabol ALTran MTNLiivak KfalseAnti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel <i>Streptococcus pyogenes</i> Cas9 variants with both increased and decreased DNA bi2025-01-01T00:00:00Z2025 Dec2026-06-14T03:13:39.218Z2026-06-14T03:08:35.092ZS-EPMC127106524141544210.64898/2025.12.11.693673