Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping

Abstract

Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype-phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Here we report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.

Fig. 1. The MAGIC pipeline for genome-wide mapping genotype–phenotype relationships.

Guide sequences for genome-scale activation (orange), interference (light blue), and deletion (magenta) were synthesized as arrayed oligos on DNA chip and cloned into the corresponding gRNA expression plasmids using Golden-Gate Assembly. The iMAGIC library was constructed by transforming the pooled plasmid libraries into the CRISPR-AID integrated yeast strain, and subject to growth enrichment under various conditions or high throughput screening. The enrichment and depletion of guide sequences were profiled using next-generation sequencing. The iMAGIC workflow can be iterated to better understand and engineer complex phenotypes.

Fig. 2. Iterative MAGIC enabled genome-wide mapping of furfural tolerance in yeast.

The iMAGIC library was subject to iterative rounds of screening under gradually increased furfural concentration, 5, 10, and 15 mM for the first a, b, second c, d, and third e, f round of iMAGIC screening, respectively. The guide sequences of the enriched libraries were profiled a, c, e using next-generation sequencing and the top hits were verified b, d, f under the corresponding screening condition. The red dots represented the control guide sequences. Orange bars represented activation targets, light blue for repression, and magenta for deletion. Error bars represent the mean ± s.d. of biological triplicates. The source data for figures b, d, and f are provided as a Source Data file.

Fig. 3. iMAGIC for the construction of a furfural tolerant yeast strain.

a Furfural tolerance of the engineered strains identified in each round of iMAGIC screening, R1, R2, and R3. The cell densities of the engineered strains were normalized to the wild-type (WT) strain under the specified conditions (red bars for 7.5 mM furfural, blue for 12.5 mM, and purple for 17.5 mM). b Verification of gain-of-function and reduction-of-function mutations by qPCR. The expression level of each target (SIZ1, NAT1, and PDR1) was compared before (NC, red) and after (INT, blue) CRISPRa or CRISPRi cassette integration. Fermentation profiles including cell density c, glucose consumption d, ethanol production e, as well as furfural and furfuryl alcohol (FfOH) concentration f of WT (black square and blue triangle) and R3 (red circle and purple diamond) in synthetic medium with (blue triangle and purple diamond) or without (black square and red circle) the supplementation of 17.5 mM furfural (Ff). A single colony of WT or R3 was inoculated into 3 mL SED/G418 medium and cultured until saturation, which was then transferred into 50 mL fresh SED/G418 medium with or without the supplementation of 17.5 mM furfural in a 250 mL un-baffled shaker flask. Fermentation was performed under oxygen-limited conditions (30 °C and 100 rpm), and samples were taken every 24 h. The decrease of furfural concentration in WT might result from evaporation, as no growth and furfuryl alcohol production were observed. Notably, the cell density (biomass accumulation) in f was determined by measuring the absorbance at 600 nm using a UV–vis spectrometer. Error bars represent the mean ± s.d. of biological triplicates. The source data are provided as a Source Data file.

Fig. 4. Synergistic interactions among iMAGIC identified targets (T).

Single (T1, T2, and T3), double (T1 + T2, T1 + T3, and T2 + T3), and triple (T1 + T2 + T3) mutants were constructed to investigate the synergistic interactions among SIZ1i, NAT1a, and/or PDR1i for enhanced tolerance against furfural with a final concentration of 7.5 mM (red), 12.5 mM (blue), and 17.5 mM (purple). Error bars represent the mean ± s.d. of biological triplicates. The source data are provided as a Source Data file.

Fig. 5. Engineering of furfural tolerance with sMAGIC.

a The sMAGIC pipeline for furfural tolerance engineering. The sMAGIC plasmid library was constructed by assembling two genome-scale CRISPR-AID (LibA + LibI + LibD) into the same vector [Lib(A + I + D)×(A + I + D)] using Golden-Gate Assembly. The sMAGIC yeast library was constructed by transforming the sMAGIC plasmid library into the bAID strain and subject to colony-size-based high throughput screening on agar plates. b Furfural tolerance of the engineered strains identified by sMAGIC screening. The strains containing an empty vector (NC) and SIZ1i-NAT1a (PC) were included as a negative control and a positive control, respectively. c Sequencing results of top clones identified by sMAGIC screening, including sMAGIC1 (UBC9i-SFH1a), sMAGIC2 (SIZ1d-SPE29i), and sMAGIC3 (SLX5i-SDS3i). d Single (UBC9i or SFH1a) and double (UBC9i-SFH1a) mutants were constructed to investigate the synergistic interactions between SFH1a and UBC9i for enhanced tolerance against furfural (10 mM). Error bars represent the mean ± s.d. of biological triplicates. The source data for figures b and d are provided as a Source Data file.