Selection of chromosomal DNA libraries using a multiplex CRISPR system

Abstract

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.DOI: http://dx.doi.org/10.7554/eLife.03703.001

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

“…Centromeric plasmids contain an autonomously replicating sequence (ARS) and a centromere (CEN) to mediate plasmid replication and segregation during cell division. On average, these plasmids are maintained with 1.2-2.5 copies/haploid genome, but the copy number varies widely among individual cells and the plasmids are lost in about 3% of cell divisions (Fang et al, 2011;Karim et al, 2013;Ryan et al, 2014;Sikorski and Hieter, 1989). Episomal plasmids bear a control sequence from the yeast endogenous 2 μ plasmid (ORI and STB) (Christianson et al, 1992).…”

Genome-Editing Technologies: Principles and Applications

“…CRISPRCas9 has been used to inactivate multiple genes in actinomycetes (Tong et al 2015), indicating its ability to enable the creation of designer bacterial strains with enhanced metabolite production capabilities. CRISPR has also facilitated multiplexed metabolic pathway engineering in yeast at high efficiencies (Jakociunas et al 2015a,b), as well as random mutagenesis of yeast chromosomal DNA for phenotypic screening of desired mutants (Ryan et al 2014). Indeed, genome-wide CRISPR-based knockout screens hold tremendous potential for functional genomics (Hilton and Gersbach 2015), having facilitated the discovery of genomic loci that confer drug resistance to cells (Koike-Yusa et al 2014;Shalem et al 2014;Wang et al 2014a;Zhou et al 2014), uncovered how cells can control induction of the host immune response (Parnas et al 2015), provided new insights into the genetic basis of cellular fitness (Hart et al 2015;Wang et al 2015b), and even shed light on how certain viruses induce cell death .…”

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…U6 mammalian promoter requires to have a G at the 5′ end of the transcript [28]. Transcriptional expression can be improved by inserting self-processing elements, such as HDV ribozyme and tRNAs, at the 5′ or 3′ end to prevent potential degradation of the transcript [49,83]. Processing elements can also be exploited to multiplex several gRNAs in a row by collocating those element between each gRNAs [17] (Fig.…”

“…For example, Shi et al specifically designed gRNAs to target multiple delta sites in the yeast genome, ultimately achieving 18-copy genomic integrations of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, in S. cerevisiae [88]. DNA libraries, such as error-prone PCRs derived or double-stranded fragments obtained from DNA synthesizing companies, can be genomically integrated to find variants of a studied enzyme with enhanced catalytic activities or optimal level of expression [64,83]. Genomically integrated DNA libraries offer several advantages compared to plasmid based strategies, especially in terms of expression stability [83].…”

“…DNA libraries, such as error-prone PCRs derived or double-stranded fragments obtained from DNA synthesizing companies, can be genomically integrated to find variants of a studied enzyme with enhanced catalytic activities or optimal level of expression [64,83]. Genomically integrated DNA libraries offer several advantages compared to plasmid based strategies, especially in terms of expression stability [83]. Liang et al used that strategy to integrate 640 ribosome binding sites (RBS) for five different enzymes involved in the production of isopropanol in E. coli [64] (Fig.…”