Genetic Engineering Publications - GEG Tech top picks

A suite of high-throughput assays enables discovery of small-molecule inhibitors of CRISPR-Cas9 that are cell permeable, and non-toxic, providing a chemical means to control SpCas9-based tools.

Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. Here the scientists describe CRISPR–Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.

Here, the scientists developed a CRISPR-library-based approach for highly efficient and precise genome-wide variant engineering. They used our method to examine the functional consequences of premature-termination codons (PTCs) at different locations within all annotated essential genes in yeast. This approach can be used to profile the effects of large classes of variants in a high-throughput manner.

Garst et al. sought to combine these strategies to engineer precise designed mutations on a genomic scale. They generated a library of >50,000 plasmids containing 3 variable but covalently coupled components: a gRNA-expressing region, a replacement cassette to generate a defined mutation at the particular gRNA target site, and a barcode sequence to uniquely identify the overall construct. As an additional feature, the replacement cassette was designed to generate the main target-site mutation, as well as a synonymous mutation in a nearby protospacer-adjacent motif (PAM) to minimize further Cas9 cleavage at edited loci.

In initial proof-of-principle tests, the team transfected single constructs or pooled libraries of constructs into Cas9-expressing Escherichia coli cells and achieved ~70% efficiency of correct editing at target loci. They then tested single-gene CREATE libraries for functional screening: mutagenesis of folA (which encodes dihydrofolate reductase) to screen for trimethoprim resistance and mutagenesis of the acrB drug efflux pump to screen for isobutanol resistance. In both cases, sequencing across the barcode region in populations of E. coli cells was used to infer genome edits that were relatively enriched in the treated samples, and follow-up validation experiments using individual constructs confirmed that these edits indeed conferred treatment resistance.