CRISPR Assissted Rational Protein Engineering: tool development for genome scale metabolic engineering

Clustered regularly interspaced short palindromic repeat (CRISPR) systems are adaptive immune systems in bacteria that utilize a short sequence of RNA to recognize invading fragments of DNA and target their destruction. The high specificity and ease with which these RNA guided endonucleases can be targeted to different unique chromosomal sites has made them an ideal tool for precision genome editing applications. We have developed a method termed CRISPR Assisted Rational Protein Engineering (CARPE) which leverages parallel DNA synthesis and CRISPR based selection to create rational libraries at high efficiency in multiplex. This technology relies on coupling programmed codon and proto-spacer adjacent motif (PAM) mutations via oligo synthesis and allows us to embed designed codon mutations across ~ 1 kb segments of the chromosome with high efficiency (30-80%) in a markerless fashion. We have utilized CARPE to create libraries of both essential and nonessential genes in the E. coli genome. The prospect of engineering chromosomes with such high precision and throughput offers an exciting new avenue for more tightly integrating future protein and metabolic engineering efforts, particularly as biological engineering efforts are extended to more complicated engineering tasks.