(563c) Highly Multiplexed CRISPR-Cas9 Applications with Extra Long Sgrna Arrays

CRISPR/Cas9 systems enable targeted genome editing and gene regulation for widespread biotechnology applications. However, only a small number of genetic loci can be targeted simultaneously, due to the difficulty of synthesizing, cloning, and expressing many repetitive crRNAs. Here, we massively expand the scalability of multiplexed CRISPR applications by demonstrating how up to 22 genetic loci can be simultaneously targeted for gene editing or gene regulation. We developed Extra Long sgRNA Arrays (ELSAs) to express many sgRNAs as independently transcribed units in a highly compact and highly non-repetitive architecture. To do this, we designed and characterized toolboxes of highly non-repetitive promoters, sgRNA handles, and terminators, combining design algorithms and machine learning to identify the largest possible toolbox of non-repetitive parts that satisfy sequence-structure-function constraints. We also design guide RNA sequences to minimize off-target activities. As a result, ELSAs are inexpensively synthesized with high success rates, stably expressed inside genomes, and capable of regulating up to 22 targeted gene expression levels. We demonstrate ELSA capabilities by massively reprogramming E. coli physiology across three applications: redirecting metabolic fluxes to over-produce a specialty chemical, creating a many-auxotrophic strain for biocontainment, and triggering susceptibility to several common antibiotics. In each application, we show how ELSAs can be rapidly synthesized, cloned, and expressed to achieve desired phenotypes.

Multiplex use of CRISPR-Cas9 allows simultaneous editing or regulation of multiple genes to achieve complex phenotypic outcomes for dozens of potential applications across the life sciences. However, difficulty with synthesis and stable expression of repetitive CRISPR arrays limit the size and applied use of these systems. To enable direct gene synthesis and stable expression of extra-long sgRNA arrays (ELSAs), we developed a toolbox of 28 high-performance, non-repetitive sgRNA handles compatible with the S. pyogenes Cas9 (SpCas9) protein. We developed an automated design algorithm to create synthesizable and robust ELSAs using non-repetitive parts. We apply the design algorithm and the non-repetitive sgRNAs toolbox to design and rapidly implement ELSAs that result in complex phenotypes in three E. coli strains by simultaneously knocking down up to 22 genes to achieve succinate overproduction, tolerance to acetate and osmotic stress conditions, auxotrophy to 10 amino acids, and increased susceptibility to 6 common antibiotics.