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High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast

Abstract

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80–100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

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Figure 1: Guide+donor genome-editing platform for engineering and phenotypically characterizing programmed mutations in pool.
Figure 2: Guide+donor library of sgs1 mutants in response to HU.
Figure 3: Guide+donor library of amino acid substitutions of selected conserved residues in SGS1 in response to various concentrations of HU.
Figure 4: smORF mutant library subjected to different phenotypic screens.

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Acknowledgements

G.M.C. was supported by NIH grants RM1 HG008525 and P50 HG005550. A.C. was funded by the National Cancer Institute grant no. 5T32CA009216-34. J.J.C. was funded by the Defense Threat Reduction Agency grant HDTRA1-14-1-0006, the Paul G. Allen Frontiers Group. Y.Y. was supported by the Damon Runyon Research Foundation grant DRG-2248-16.

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Authors

Contributions

X.G. and A.C. conceived the idea, led the study, and designed all experiments. A.C. and R.C. with input from J.E.D. demonstrated the initial feasibility of the guide+donor approach. X.G. performed majority of the experiments, including the oligo library design, library construction and analysis, with significant technical contribution from A.T. Y.C. provided expertise in statistical analysis. Y.Y. performed the whole genome sequencing experiment for off-target analysis. C.K. generated the RNA-seq data for the BY4741 yeast strain, provided the FPKM values and analyzed the whole genome data from yeast isolates modified by guide+donor for off-target effects. S.L.G. assisted with oligo library design. E.K. provided insight with regard to library construction methods and analysis. M.S. provided technical expertise with regard to methods to increase guide+donor efficiency. J.J.C. and G.M.C. oversaw the study. X.G. and A.C. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Alejandro Chavez or George M Church.

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Competing interests

G.M.C. is the founder and holds leadership positions in many companies (http://arep.med.harvard.edu/gmc/tech.html). X.G., A.C., M.S., and E.K. have filed a patent application (US Patent Application 62/348,438) relating to this work.

Integrated supplementary information

Supplementary Figure 1 Enhanced transformation efficiency with engineered guide+donor approach.

a Schematic representations of unmodified guide+donor configuration (circular) and our engineered version (linearized) along with their corresponding transformation efficiencies. SNR52 promoter, N20, donor, and URA3 marker are labeled. Imaged plates are representative of 34 guide+donor yeast transformations in b targeting 34 different genomic sites performed with both configurations. b Scatterplot comparing transformation efficiencies of the selected 34 guide+donor plasmids between the unmodified and the engineered approaches in yeast strains expressing (blue) and not expressing Cas9 (orange). Genomic target corresponds to each guide+donor is indicated. Each guide+donor yeast transformation was performed twice. Relevant genotype of yeast strain background is exhibited on the x-axis. Average fold change in transformation efficiency is indicated on the y-axis and was calculated based on the number of transformants obtained from the engineered linearized plasmid setup divided by the number of transformants obtained from the circular plasmid transformation.

Supplementary Figure 2 Guide+donor editing efficiency of SNPs at various distances from the cut site.

Frequency of correct SNP edits are displayed on the y-axis with varying distances from the Cas9-generated cut site indicated on the x-axis. Position of cut site is marked by the middle vertical line (red). Number of samples genotyped are indicated.

Supplementary Figure 3 Examining the influence of homology lengths on transformation efficiency of SNPs further away from the PAM.

a Schematic representation of a region of ADE2 centered around an N20-targeting site with SNPs (blue), SNPs distances, various homology lengths, cut site position, and wildtype and mutated PAMs (red) indicated. b Transformation efficiency of ADE2 guide+donors with varying donor homology lengths and SNPs distances from PAM as indicated on the legend. Transformation efficiency was calculated by the number of colonies obtained in the presence of Cas9 divided by the number of colonies in the absence of Cas9. Average transformation efficiency is displayed on the y-axis (n=2 independent cultures).

Supplementary Figure 4 Testing different design parameters for efficient programmed genomic edits at the ADE2 locus.

a Frequency of correct programmed deletions are indicated on the y-axis with increasing sizes of deletions labeled on the x-axis. b Frequency of correct edits on the y-axis with replacement of a 61bp endogenous sequence region by increasing sizes of a linker sequence as denoted on the x-axis.

Supplementary Figure 5 Whole genome sequencing of guide+donor-edited yeast strains to determine off-target effects.

Whole genome sequence alignment of strains transformed with guide+donors targeting (a) ADE2 to delete a w61bp region, (b) SGS1 to delete a 60bp fragment, or (c) 3bp ATG start site.

Supplementary Figure 6 Construction and functional interrogation of guide+donor library targeting C-terminus of SGS1 with controls targeting ARS214.

a Graphical representation of programmed edits in ARS214 (grey) and SGS1 (blue) targeting C-terminus generated by guide+donor constructs followed by phenotypic characterization in b and c. Asterisk, dotted box, and solid dash denote substitution, deletion, and replacement of an amino acid stretch with a linker sequence, respectively. Figures not drawn to scale. b Dot plot showing fold change of guide+donor constructs targeting ARS214 and SGS1 in response to HU condition. Types of genetic modifications are labeled on the x-axis. Depletion is represented in log2 scale on the y-axis and compared between ARS214 and SGS1. Yeast library transformation was performed twice. c Dot plot showing depletion of ARS214 and SGS1 guide+donor constructs in mms4Δ genetic background. Genetic modifications and their corresponding abundance in log2 scale are indicated on x- and y-axes, respectively. Two independent yeast library transformations were transformed.

Supplementary Figure 7 Phenotypic testing of individual guide+donor generated sgs1 protein variants in response to HU.

Cells were grown to log phase. 3μl of each undiluted and 5-fold serially diluted culture were spotted onto control SC-URA plate or SC-URA plate containing 40mM HU. All plates were incubated at 30ºC for 48 hours and photographed.

Supplementary Figure 8 Phenotypic validation of individual mutants from sgs1 amino acid library screen.

Wildtype and selected sensitive and non-sensitive hits from HU library screen are indicated. 3μl of each undiluted and 5-fold serially diluted culture were spotted onto control SC-URA plate or SC-URA plate containing 40mM HU. All plates were incubated at 30ºC for 48 hours and photographed.

Supplementary Figure 9 Replicate analysis of smORF library screens in response to various test conditions.

Replicate analysis of log2 fold changes between biological replicates of two independent yeast transformations for each smORF library screen in Figure 4 (a-d). Pearson correlation coefficient is indicated.

Supplementary Figure 10 Phenotypic validation of smORF mutants from heat and fluconazole screens.

Wildtype and selected sensitive and non-sensitive hits from smORF library screens are indicated. For the heat screen validation (a), cells were grown to log phase. 3μl of each undiluted and 5-fold serially diluted culture were spotted onto SC-URA plates that were incubated at the indicated temperatures for 48 hours and photographed. For the fluconazole screen validation (b), cells were cultured in a similar manner followed by spotting 5μl of each undiluted and 5-fold serially diluted culture onto SC-URA plates with and without fluconazole. Plates were incubated at 30ºC for 48 hours and photographed.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 953 kb)

Life Sciences Reporting Summary (PDF 132 kb)

Supplementary Tables

Supplementary tables1–7 (PDF 273 kb)

Supplementary Data 1

Log2-fold changes in abundance of guide+donor members in SGS1 tiling deletion screen (XLSX 60 kb)

Supplementary Data 2

Log2-fold changes in abundance of guide+donor members in Sgs1 conserved residue amino acid substitution screen (XLSX 49 kb)

Supplementary Data 3

Log2-fold changes in abundance of guide+donor members in smORF screen (XLSX 404 kb)

Supplementary Data 4

Analysis of amino acid length, gene expression, prediction of secondary structure formation and level of conservation in humans between smORFs and ORFs (XLSX 45 kb)

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Guo, X., Chavez, A., Tung, A. et al. High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast. Nat Biotechnol 36, 540–546 (2018). https://doi.org/10.1038/nbt.4147

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