Advances in DNA synthesis and sequencing have motivated increasingly complex efforts for programming cells on laboratory timescales. Realization of such efforts requires strategies that span the design-build-test forward-engineering cycle by not only precisely generating large numbers of mutant designs but also mapping such designs to targeted traits at similar throughputs. Here we describe CRISPR EnAbled Trackable genome Engineering (CREATE), a technology that couples the high efficiency of CRISPR editing with the massively multiplexed rational design offered by parallel oligomer synthesis. CREATE enables generation of tens of thousands of designer genome edits and simultaneous sequence-activity mapping of each synthetic genotype. We demonstrate the broad utility of CREATE for forward genome engineering by the simultaneous mapping of sequence-activity relationships for single proteins, genome wide mutations found in adaptive laboratory evolution experiments and directed evolution of multi-protein pathways and complexes and regulatory networks. Owing to the fact that CREATE is based on oligo-scale synthetic DNA ensures that the design, construction, and mapping of mutations of various types (SNP, indels, etc.) should directly scale with improving throughput and cost of DNA synthesis and will provide an economical approach to rational genome scale engineering efforts.

Trackable genome engineering with single nucleotide resolution

251st National Meeting (2016)

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