A novel CRISPR based approach to trackable multiplexed genome scale engineering

Genetically engineered organisms play a central role in the production of foods, chemicals and pharmaceuticals in the modern global economy. Although decades of research and directed evolution have derived many interesting phenotypes the challenge of developing rapid genome scale engineering technologies that enable a more forward design-build-test engineering cycle remain. Highthroughput genomic prototyping and sequence to activity mapping requires not only flexible, precise and efficient methods for generating large mutant libraries but also requires that these genotypes can be readily tracked on a population wide scale. This talk will describe CRISPR EnAbled Trackable genome Engineering (CREATE), a strategy that couples the high efficiency of CRISPR editing with massively parallel oligomer synthesis to enable such a task. To demonstrate the broad utility of CREATE for trackable editing we demonstrate mapping of over 50,000 sequence-activity relationships under a variety of selective pressures and show that the engineering capabilities can can readily scale from single proteins to genome wide targeting of promoters or user specified open reading frames. This technology was applied to identification and validation of multiple novel and previously validated mutations that confer tolerance to a variety of industrially relevant compounds including isobutanol, furfural, and common antibiotics.