Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering

Abstract

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9

“…Finally, at present, most of the CRISPR/Cas9-based directed evolution methods rely on integration of relative small (80-120 bp) DNA fragments (Barbieri et al, 2017;Garst et al, 2017;Guo et al, 2018) and may require time-consuming construction of donor variant libraries and/or relative costly DNA synthesis of diversified array-based oligos (Nyerges et al, 2018;Roy et al, 2018). With CasPER, multiplex genome engineering of larger genomic loci is now demonstrated at very high efficiencies along the full size of donor fragment lengths, and should thereby enable cost-effective, high-throughput and robust evolution studies of even complex multi-genic traits in any organism supporting genome integration of heterologous DNA by homologous recombination.…”

“…Cas9 is an RNA-guided endonuclease that introduces double-stranded breaks (DSBs) at almost any target DNA locus where a protospacer-adjacent motif (PAM; NGG) is present, yielding several hundred thousand genomic target sites in an average eukaryotic genome (DiCarlo et al, 2013). With the ability to target and integrate heterologous DNA fragments at high efficiencies by way of homologous recombination, several studies have investigated directed evolution and sequence-function relationships of regulatory and genic regions in genomic contexts, albeit using short oligonucleotides as repair donors (Barbieri et al, 2017;Findlay et al, 2014;Garst et al, 2017;Storici et al, 2001;Wang et al, 2009). In addition, larger mutagenized DNA fragments were in vivo assembled and integrated into the S. cerevisiae genome by employing Cas9 and a selection marker (Ryan et al, 2014).…”

High-throughput creation and functional profiling of eukaryotic DNA sequence variant libraries using CRISPR/Cas9

“…The advantages of our concatenated guide+donor design are threefold: a) It enables rapid cloning of all library members within a single set of reactions, b) it allows simultaneous delivery of both the guide and donor in one contiguous unit thus preventing uncoupling that may result in inefficient repair and unproductive repair outcomes, and c) it enables high-throughput molecular phenotyping using next generation (NGS) sequencing with each guide+donor containing a plasmid serving as unique barcodes for tracking edited cells. A similar concept of in cis delivery of guide+donor was recently demonstrated in bacteria 4 .…”

In situ genotyping of a pooled strain library after characterizing complex phenotypes

“…For example, one can make pooled dCas9 libraries based on plasmids harboring both a genotype-identifying barcode and a sgRNA gene (Dixit et al, 2016;Jaitin et al, 2016;Peters et al, 2016;Garst et al, 2017;Otoupal et al, 2017) for labeling genetic loci (Chen et al, 2013) or knocking down/activating genes throughout the chromosome. Alternatively, pooled A Examples of channels and cells in the custom-made microfluidic device which are imaged in both phase contrast (top) and fluorescence microscopy (bottom).…”