bioRxiv : the preprint server for biologyDec 19, 2025

Using anti-CRISPR to continuously improve CRISPR-Cas9 gene editing in human cells

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

CRISPR-MACE enabled continuous evolution of Cas9 variants directly in human cells.

Evidence

This human-cell platform experiment combined virus-based continuous evolution with anti-CRISPR-tuned selection to evolve Streptococcus pyogenes Cas9 variants with altered DNA binding and nearly 1000-fold increased AcrIIA4 resistance.

Caveat

The work establishes an engineering platform and evolved Cas9 properties in human cells, but the abstract does not show therapeutic performance or in vivo editing outcomes.

Simplified

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 novelCas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells. Streptococcus pyogenes