The development of novel CRISPR–Cas genome editing tools continues to drive significant advances in the life sciences. Particularly, compact CRISPR–Cas such as Cas12f (composed of 400-600 amino acids) or Cas12j (composed of 700-800 amino acids) are promising candidates. Nevertheless, these CRISPR–Cas systems are hampered by their limited editing efficiencies in eukaryotic cells. To pave the way for broader applications in basic research and therapeutic applications, it is important to improve their editing capabilities, specificity and expand their targeting scope.

In this study, we introduce the concept of CRISPR associated substrate-linked directed evolution (CaSLiDE) as a method to evolve CRISPR-Cas systems with increased efficiency and precision. We applied the CaSLiDE approach to evolve different Cas12f proteins, the ABE8e TadA domain and miniature base editors, thereby improving their utility. Our findings compellingly illustrate the potency of the CaSLiDE approach in the directed evolution of genome editing enzymes, underscoring its significance for future applications.