Using Anti-CRISPR to Continuously Improve CRISPR-Cas9 Function in Human Cells

CRISPR-MACE enables nearly 1,000-fold enhanced resistance to the strongest known inhibitor of SpCas9.

• CRISPR-MACE combines virus-based continuous evolution with anti-CRISPR selection in human cells.
• Cas9 variants generated through this method show both increased and decreased DNA binding capacity.
• A specific mutation in Cas9 consistently appears early in independent evolution campaigns.
• The approach provides a framework for continuous evolution of genome-targeting agents directly within mammalian cells.
• Key principles and synthetic circuits for evolving CRISPR-Cas systems are established through this work.

Simplified