(A) Schematic depicts the generation of T4del. The clamp-loader locus in wild-type T4 bacteriophage, containing genes for the sliding clamp and the ATPase and clasp subunits of the clamp loader, is replaced with a CRISPR-cas12 target site. See methods for details. (B) In the high-throughput phage-propagation assay, T4del infects bacteria carrying (i) a plasmid-encoded CRISPR-cas12 (not shown) programmed to target the recombination site inserted in A and (ii) a plasmid containing variant genes of the clamp-loader locus. Upon infection, the clamp-loader locus recombines into the T4del genome, and this genome is replicated by the variant of the clamp and clamp-loader genes present in each cell. Variants with reduction in function in the clamp-loader activity will produce fewer phage particles relative to the variants with wildtype-like activity. (C) Workflow for comprehensive assessment of fitness effects of all possible single amino-acid mutants of the sliding clamp and the clamp-loader subunits. Codons corresponding to each amino acid are individually mutagenized to NNS (N is a mixture of all four nucleotide bases, S is a mixture of G and C) by PCR, combined in equimolar ratios and transformed into E. coli for cloning.