CRI-SPA: a high-throughput method for systematic genetic editing of yeast libraries

Abstract

Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.

Graphical Abstract

Figure 1.

The CRI-SPA gene-transfer system. (A) Left: The CRI-SPA Donor (CD) strain and the recipient library strains (here YKO). Right: Graphics of the individual genetic components are shown in the legend box. The YKO strains contain a specific deletion marked by the antibiotic marker 1 (AB1) and a target site for the Cas9-sgRNA CRISPR nuclease. The CD strains contain: Kl_URA3-GAL1p cassettes at the centromeres of all chromosomes, a cas9 gene, a selectable CRI-SPA plasmid maintained by antibiotic marker (AB2) and encoding a sgRNA targeting the insertion site of the genetic feature (blocked in the CD), a genetic feature of interest coupled to an antibiotic marker, AB3 (optional), and a Kl_URA3 gene between the genetic feature of interest and the telomere. (B) The individual steps of the CRI-SPA procedure (see main text for details). Step 1, a CD strain is pinned onto all strains of the library plates and incubated for mating. Step 2, diploids are exhausted for glucose via growth on raffinose and selected for the marker in the library strains (AB1) and the marker on the CRI-SPA plasmid (AB2). In the diploid stage, the target site in the recipient strain is cleaved by Cas9-sgRNA. Repair of the resulting DNA DSB by gene conversion using the corresponding donor site in the CD strain as template transfers the genetic feature to the recipient locus. Note that the target site of the Cas9-sgRNA in the recipient chromosome is destroyed by insertion of the genetic feature of interest. Step 3, galactose induced SPA eliminates the donor chromosomes. Step 4, haploid cells containing only recipient chromosomes are selected on 5-FOA. Step 5, the modified library strains are obtained by selecting for AB1, which marks the YKO deletion, and optionally, by selecting for the genetic feature (AB3).

Figure 2.

Transfer of the ade2Δ::hphNT1 cassette from a CD to plate 9 of the YKO library. CRI-SPA mediated transfer of the ade2Δ::hphNT1 cassette to the mutant strains of plate 9 of the YKO library was performed in quadruplicate. The final CRI-SPA plate was photographed and shown in the middle. Selection applied at the different CRI-SPA steps are indicated to the left by the color code. G418 selects for the kanMX marker in the gene deletion cassettes of the YKO library, NTC selects for the CRI-SPA plasmid, HYG selects for the ADE2 disruption cassette ade2Δ::hphNT1, and 5-FOA counter-select donor strain chromosomes, which all contain Kl_URA3 markers. The four ade2Δ::hphNT1 scp160Δ::kanMX and the four ade2Δ::hphNT1 ade3Δ::kanMX CRI-SPA colonies are framed by blue squares. Magnifications of the ade2Δ::hphNT1 ade3Δ::kanMX quadruplets are shown to the right as indicated.

Figure 3.

Betaxanthin production in YKO strains. (A) Left: images of BY-Ref and BY-Btx colonies in visible light (top) and corresponding filtered images (bottom). Right: quantification of yellowness of BY-Ref and BY-Btx colonies by image analysis, see main text and Supplementary Figure S2 for details. (B) Left: image of BY-Ref and the BY-Btx strains in liquid SC medium. Right: quantification of fluorescence produced by BY-Ref and the BY-Btx strains in liquid SC medium. (C) Final plate of a CRI-SPA Btx-cassette transfer experiment. At the top, a scheme illustrating the Btx-cassette and how it is transferred from the CD strain to a library recipient strain. To the left, diagrams with color codes indicating selection applied at the different CRI-SPA steps. G418 selects for the kanMX marker in the gene-deletion cassettes of the YKO library, HYG selects for the CRI-SPA plasmid, NTC selects for the Btx-cassette, and 5-FOA counter-select donor strain chromosomes, which all contain Kl_URA3 markers. (D) In the middle, yellow intensity ranking of all YKO mutant strains after CRI-SPA mediated Btx-cassette transfer. To the left, mosaic images of the four individual replicates obtained with selected YKO mutants displaying higher, similar, or lower yellowness as compared to BY-Btx. Mutants in red frames were purified and restreaked on solid YPD medium, see images to the right.

Figure 4.

Reproducibility of betaxanthin CRI-SPA experiments in the presence and absence of pathway transfer selection. (A) The mean yellow color intensities obtained for each mutant in repeat 1 and repeat 2, which both were performed in the presence of natMX to select for the Btx-cassette genes. (B) The mean yellow color intensities obtained for each mutant in trial 1 and trial 3. Trial 3 was performed in the absence of natMX. The top and bottom 192 genes and their overlap across screens are colored and displayed in flanking Venn diagrams. Insets show the correlation for colony fitness across screens.

Figure 5

Betaxanthin yield and fitness relationship reveals mechanistic patterns in betaxanthin production. Colony size (i.e. fitness) vs Yellow Color intensity (i.e. Betaxanthin Yield). The results of Gene Ontology Enrichment Analyses on several subgroups of genes are shown as graphs for the bottom hits (white, 1.2 std below the screen mean) and top hits (yellow, 1.2 std above the screen mean). As indicated by the bottom right scale, node size indicates the significance of GO terms enrichment, edge transparency indicates the number of shared genes between GO terms. The cyan group did not show any term enrichment. The position of the BY-Btx strain is marked with a black asterisk.

Figure 6.

CRI-SPA hits identified on solid medium produce enhanced amounts of betaxanthin in liquid medium. (A) selected Btx-hit strains yellow and fitness scores within the screen. (B) Correlation between CRI-SPA yellowness score and fluorescence of reversed engineered hits after 7 days cultivation in liquid SC medium. Error bars show standard deviations for 16 and 3 replicates for CRI-SPA and liquid Fluorescence, respectively. Markers coloring scheme, below inset, is defined by the agreement between CRI-SPA score and fluorescence in liquid medium, and significance difference from WT reported by a one-sided Welch's test.