A Crispr Based Approach for Trackable Multiplexed Engineering on a Genome Scale

Advances in DNA synthesis and sequencing have motivated increasing efforts to program cells on laboratory timescales. Realization of such efforts requires strategies that span the design-build-test forward-engineering cycle by not only precisely and efficiently generating large numbers of mutant designs but also by mapping the effects of these mutations at similar throughputs. While CRISPR based methods for genome-editing are extremely efficient, strategies for parallel editing at sites located throughout a genome have been limited to the study of gene knockouts, preventing systematic analysis at single nucleotide or amino acid level. Here we describe CRISPR EnAbled Trackable genome Engineering (CREATE), a strategy that couples the high efficiency of CRISPR editing with massively parallel oligomer synthesis to perform precision editing on a genome wide scale. In addition to generating genomic diversity, CREATE provides a simple method for tracking each genotype in the population, thus enabling rapid assessment of mutational fitness under selective growth conditions. We demonstrate the broad utility of CREATE for genome engineering applications by mapping of over 50,000 sequence-activity relationships in a variety of contexts. CREATE can readily scale the procedure of systematic mutagenesis of single genes up to genome wide promoter and open reading frame engineering. As proof of concept we demonstrate this approach for the rapid identification and validation of novel antibiotic resistance and solvent tolerance mutations in E. coli.