PROJECT SUMMARY Genome editing technologies have catalyzed major advances across basic and applied biomedical research fields. Although the adaptation of CRISPR-Cas enzymes for genome editing has facilitated and dramatically accelerated the ability to edit nucleic acid sequences in living cells, many genetic engineering approaches are performed using a single enzyme that has notable limitations. The naturally occurring CRISPR-Cas9 effector from the bacterium Streptococcus pyogenes (SpCas9) can function efficiently for certain editing applications, but is not a ‘one-size-fits-all’ solution for treating the diversity of sequences that cause genetic disorders. The clinical potential of SpCas9 is inherently limited due to the natural characteristics of the enzyme, including a requirement to bind a short DNA motif to initiate editing. This motif only occurs in a fraction of the genome, preventing SpCas9 from editing many disease-causing mutations that do not harbor this sequence. The inability of SpCas9 to target a broad range of DNA sites, along with other suboptimal characteristics, illustrates the need for innovations to unlock the wide potential of genome editing in the clinic. One solution to enable more comprehensive genome targeting is to utilize directed evolution to engineer new forms of SpCas9 that can recognize new motifs. However, traditional protein engineering approaches remain low-throughput, costly, and laborious. Here we will close these technological and methodological gaps by optimizing scalable methods to engineer and characterize novel Cas variants with improved properties. Our proposed research will address prominent limitations of CRISPR-Cas enzymes by: (1) developing scalable experimental approaches to more rapidly and effectively engineer and characterize proteins, (2) optimizing machine learning-guided directed evolution to create a catalog of customizable DNA editors, and (3) as proof-of-concept, thoroughly evaluate a catalog of PAM-selective editors against mutations that cause common and rare diseases. The long-term vision of this project is to create a catalog of bespoke editors that together can systematically target the genome without sacrificing other important properties like specificity. To fully democratize editing, this collection of optimized CRISPR technologies will create a virtual ‘one-stop-shop’ for researchers and clinicians seeking optimized genetic treatments for patients. Successful completion of the proposed studies will synergize experimental and computational methods, will provide novel scalable approaches for characterizing and improving the activities of genome editing technologies, and will exponentially expand the capabilities within the editing ‘toolbox’. Together, the development and implementation of a catalog of custom Cas editors and will accelerate and create a blueprint for the translation of safe and effective CRISPR therapies to benefit patients.