CATS: Cas9-assisted tag switching. A high-throughput method for exchanging genomic peptide tags in yeast

Abstract

Background: The creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library.

Results: We present a new recombination-based method, CATS - Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining.

Conclusions: The method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.

Keywords: Array; CRISPR-Cas9; GFP collection; SPA; Tag switching; Yeast.

Fig. 1

CRISPR-induced mutation frequency in CAN1 and plasmid loss assays. a Frequency of mutations in the CAN1 gene assessed by the formation of colonies on plates containing canavanine, which is toxic to CAN1⁺ yeast. The plasmids in strain PT141 are specified on the x-axis, and each column is a single experiment. Frequencies were calculated from the number of colonies on canavanine-containing plates compared with no drug (the media lacked uracil and leucine to select for both plasmids and also arginine to allow canavanine toxicity). b The rates of plasmid loss were measured, since the endonuclease complex is encoded on two separate plasmids: pCas9 and pCAN1-guide. Yeast cells (TEF1-GFP from the GFP collection) containing both plasmids were grown overnight with selection for both plasmids and then 500 cells were plated on medium that selects for the pCas9 plasmid (−leucine), the pCAN1-guide plasmid (−uracil) or both (−lecuine, −uracil). The resulting colonies were compared with growth without selection. The overnight growth medium contained either glucose (blue bars) or galactose (orange bars), the latter medium induces expression of the Cas9 gene. At least one of the plasmids, pCas9 or pCAN1-guide, was lost from 10 to 40% of cells pre-grown glucose medium. This loss rate increased to nearly 100% of cells, when the Cas9 gene was induced with galactose. The pCAN1-guide plasmid is lost more readily than the pCas9 plasmid. To determine if this loss rate was caused by the endonuclease activity, we repeated the experiment with a dead version of Cas9, which contains D10A and H840A substitutions. The plasmid loss rate on galactose was much less (30–50%) with an inactive Cas9 compared with the active Cas9, indicating that plasmid loss is associated with endonuclease function. Error bars represent exact binomial 95% confidence intervals. c The number of colonies observed on YPD (blue bars), −ARG (light green bars) and -ARG NAT (dark green bars, this media selects for the plasmid) media following galactose induction of a single plasmid encoding expression of both the Cas9 and guide targeting CAN1, and two control plasmids. Five hundred cells were plated and each column is a single experiment. d The frequency of mutations in the CAN1 gene were assessed by comparing the formation of colonies on canavanine plates, with those containing no drug. The plasmids present in strain PT141 are specified on the x-axis, and each column is a single experiment. Frequencies were calculated both without selection for the plasmid (light green bars) and with NAT selection (dark green bars). e The effect upon viability of the different endonuclease plasmids was assessed by counting the viability of cells. One thousand five hundred HTB2-GFP cells (from the GFP library) that had been transformed with the plasmids stated on the x-axis were plated onto glucose (dark green bars, CAS9 expression OFF) and galactose media (orange bars, CAS9 expression ON), in 3 replicates (4500 cells in total). Bars represent mean and error bars represent standard deviation. **, P < 0.01, *, P < 0.05, n.s., non-significant; Welch’s two-sample t-test performed on 3 replicates

Fig. 2

Schematic of the CATS method. CRISPR-Cas9 cleavage induces homologous repair. An SNR52 promoter-driven RNA guide and a GAL-L promoter-driven CAS9 sequence are contained in a single endonuclease plasmid conferring NAT resistance. A template plasmid, with a URA3 marker, contains a sequence encoding a new tag and promoter-driven marker flanked by homology to the 3′ and 5′ ends of the GFP ORF. This template plasmid contains at either end a protospacer and corresponding PAM sequence, matching that cleaved by the expressed endonuclease. Upon galactose induction, both the genomic GFP ORF and the two sites in the template plasmid will be cleaved by the Cas9 endonuclease as indicated with the scissor icon. DSB-induced repair then can replace the GFP tag with the new template sequence

Fig. 3

Testing three media transfer sequences for efficiency of incorporation of the template DNA. a Three sequences of media transfer tested on the GFP collection strain Htb2-GFP following transformation with the plasmids indicated in (b). Cells were washed twice with water between each media transfer, and incubation periods at each step are indicated. b Colonies were counted following plating of fixed numbers of cells onto SC 5-FOA and SC 5-FOA G418. Proportions represent the number of colonies formed on SC 5-FOA G418 compared to SC 5-FOA, indicating that they have integrated the RFP template plasmid, which confers G418 resistance. Results from each of the three methods in (a) are shown. The mean of 3 biological replicates is shown for each method and error bars represent standard deviation

Fig. 4

Outline of SPA-based methods for high-throughput transformation of plasmids into strains and subsequent genome editing. a Summary of the media transfer steps used for converting the Htb2 and Rpa49 GFP strains to the RFP template plasmid. Plasmids were pre-transformed into the UDS strain, which was mated with the Htb2 and Rpa49 strains from the GFP collection on YP-Raffinose, in the first step indicated. Subsequent media transfers select for diploid cells with both plasmids, then activate the GAL promoter-driven endonuclease, thereby beginning the replacement of the GFP tag with the template DNA. Indicated timescales refer to incubation times before transfer to the next media type. b High-throughput SPA method. Flowcharts indicate media transfers and incubation times on each media for trial A, which were then modified for trial B. The UDS containing the endonuclease and template plasmids was mated on YP-Raffinose with colonies from the GFP library. Selection for diploids with both plasmids was applied using -HIS G418 NAT Raffinose, before cells were transferred to galactose-containing media. This galactose induction serves two purposes: expression of the gene encoding Cas9 from the endonuclease plasmid, and selection against the UDS chromosomes through Gal-promoter mediated disruption of centromeres. Subsequent 5-FOA steps further select against the UDS chromosomes, and also against the URA3-containing template plasmid. The resulting colonies forming on Galactose 5-FOA G418 medium should therefore have a haploid karyotype of chromosomes originating from the GFP strains with the template DNA integrated. These strains were transferred to YPD G418 as a final selection step

Fig. 5

Phenotypic results from high-throughput conversion of GFP strains to RFP-G418 strains. Targeting results are indicated here for 68 strains in which a GFP signal was visible in the starting strain. Trial A and Trial B are distinguished by the separate methods indicated in Fig. 4b. The conversion from GFP to RFP is indicated in all columns by the red color. Trial A indicates microscopy results for all strains except the three indicted in black which did not form a colony on G418. Strains indicated in grey we were unable to unequivocally screen using microscopy and the remainder showed a phenotype consistent with RFP, GFP or a mixed population of both, as specified. Further analysis was then undertaken on some of the strains in Trial A, where secondary microscopy checks were performed on cells from a mixed-population colony and from a single clonal colony. The clonal colonies were then also checked for insertion of the template DNA by PCR, results indicate the detected genotype. Secondary testing for growth on 5-FOA plates shows two strains with a URA+ phenotype, indicating that the template plasmid has not been effectively counter-selected against. The methodology for Trial B was adapted to counteract this. In Trial B, cells from a mixed colony were checked with microscopy as in Trial A, then cells from mixed populations were checked again. Where possible, 3 clonal colonies were then assessed by fluorescence microscopy and PCR. The observations from each of the three colonies are indicated

Fig. 6

G418 resistance does not segregate correctly following meiosis in strains that retained their GFP signal. Two of the G418+ strains that retained their GFP signal (NUP170- and BIR1-tag) were mated with a wild-type unlabeled strain to form diploids alongside one mixed RFP/GFP strain (MPS1-tag) and one strain that had successfully converted to RFP (HTB2-RFP). The diploids were sporulated and tetrads were dissected on YPD, each tetrad was horizontally arranged and replica-plated to -HIS and YPD G418 media, then scored for growth as indicated in Supplementary Table 4. Images show the subsequent colony formation of replicated spore colonies

Fig. 7

Conversion to different fluorescent tags using variants of the template plasmid. The strain encoding Htb2-GFP was tested with 4 different template construct plasmids, specified for each horizontal panel, each encoding a different fluorescent tag combined with ADH1 promoter-driven Kanamycin. Strains were then checked with fluorescence microscopy, and signals in the DIC, RFP, Azurite, CFP and YFP channels are shown. Scale bars represent 5 μm. GFP appears in both the CFP and YFP channels due to the use of a single band pass filter. It remains distinguishable from the CFP and YFP signals however as each of these appear in only one channel

Fig. 8

Simultaneous conversion to dual targeting constructs using two endonucleases. a Schematic of the targeting process with two different DNA templates, to replace two regions of the genome. The endonuclease plasmid contains a GAL-L promoter-driven CAS9 gene and two SNR52 promoter-driven sgRNA sequences, one targeting GFP and the other CAN1. The dual-template plasmid contains two template sequences, one to replace CAN1 with HYG, and the other to replace GFP with an RFP-ADH1 promoter-Kanamycin sequence, as demonstrated in Fig. 2. Each is flanked by homology to the endogenous gene, and by the relevant protospacers, so that each endonuclease complex will cut both the genome and the plasmid template sequence, inducing HDR into both loci. b Proportions of converted strains from the dual targeting experiment illustrated in (a). CAN1 targeting confers HYG resistance, and conversion from GFP to RFP confers G418 resistance. The strain Htb2-GFP contained the dual-template plasmid alongside an endonuclease plasmid encoding both the GFP and CAN1 guides, or each of these individually, as indicated. The proportions are calculated against the number of colonies growing on YPD without selection, and shown here is the mean and standard deviation (error bars) of 3 biological replicates