Methods

Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae
AsCas12a Molecular Biology SaCas9

Yeast strains

Yeast strains used in this study are listed in Table S1. All strains are derived from Saccharomyces cerevisiae BY4741 or BY4742 (Brachmann et al. 1998). Standard culture media were used in this study (Guthrie and Fink 1991). Conventional gene deletion was performed using a PCR-based method (Longtine et al. 1998). Plasmids used for yeast strain construction are listed in Table S2.

Construction of backbone vectors for genome editing

ODNs used in this study are listed in Table S3. All ODNs for plasmid construction were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Eurofins Genomics K. K. (Tokyo, Japan). The four backbone vectors for genome editing were constructed using the seamless cloning with HiFi DNA Assembly (E2621, New England Biolabs, Ipswich, MA, USA). Restriction enzymes used for plasmid construction were purchased from New England Biolabs. PCR fragments used for plasmid construction were amplified by Q5 DNA polymerase (M0491, New England Biolabs) according to manufacturer’s instruction. Escherichia coli competent cells NEB 5-alpha (C2987, New England Biolabs), NEB Stable (C3040, New England Biolabs), or Champion DH5α high (CC5202, SMOBIO Technology, Hsinchu City, Taiwan) were used for transformation to amplify and extract plasmids. Plasmids were extracted by FastGene Plasmid Mini Kit (FG-90502, Nippon Genetics, Tokyo, Japan). Plasmids used in this study are listed in Table S2. The DNA sequence files of the backbone vectors for genome editing are available on our repository at GitHub (https://github.com/poccopen/Genome_editing_plasmid_for_budding_yeast).

Selection of target sequences for genome editing

For the insertion of mNeonGreen-encoding sequence into the CSE4 gene, we selected target sequences from the region encoding the unstructured N-terminal loop of Cse4 protein (Zhou et al. 2011; Yan et al. 2019). For the insertion of mScarlet-I-encoding sequence into the CDC3 gene, we first performed secondary structure prediction by JPred4 (Drozdetskiy et al. 2015) of Cdc3 protein and then selected target sequences from the region encoding the N-terminal region with no predicted secondary structure.

For designing single guide RNAs (sgRNAs) for SpCas9 and SaCas9, CRISPRdirect (Naito et al. 2015) was used to select target sequences. For designing CRISPR RNAs (crRNAs) for enAsCas12a, CRISPOR (Concordet and Haeussler 2018) was used to select target sequences. Target sequences for genome editing used in this study are listed in Table S4.

Construction of genome-editing plasmids

All genome-editing plasmids were constructed using the seamless cloning with Golden Gate Assembly using NEB Golden Gate Assembly Kit (BsaI-HF v2) (E1601, New England Biolabs). The ODNs for Golden Gate Assembly were automatically designed with an in-house software.

Yeast transformation for genome editing

Yeast transformation was carried out as described previously (Gietz and Woods 2002) with slight modifications. Yeast cells were cultured overnight in 2 mL of YPAD liquid medium (10 g/L Bacto Yeast Extract, #212750, Thermo Fisher Scientific, Waltham, MA, USA; 20 g/L Bacto Peptone, #211677, Thermo Fisher Scientific;100 mg/L adenine sulfate, #01990-94, Nacalai tesque, Kyoto, Japan; and 20 g/L glucose, Nacalai tesque) at 25°C with shaking at 250 rpm. The 2-mL overnight culture was centrifugated and the supernatant was removed. The cell pellet was resuspended in 0.5 mL of 0.1 M lithium acetate solution (#127-01545, FUJIFILM Wako Chemicals, Osaka, Japan). The cell suspension was incubated at 30°C for 30 min. Fifty microliters of cell suspension were thoroughly mixed with 50 μL of 1 M lithium acetate, 50 μL of 1 M dithiothreitol (#14128-04, Nacalai tesque), 5 μL of Yeastmaker Carrier DNA (10 mg/mL, #630440, Takara Bio, Kusatsu, Japan), 1 μL of genome-editing plasmid (200–600 ng), 45 μL of PCR-generated donor fragment for gene fragment insertion (1–10 μg, typically 5 μg) or ORF deletion (2.5 μg), and 300 μL of polyethylene glycol 4000 (#11574-15, Nacalai tesque). PCR fragments were amplified by Q5 DNA polymerase (New England Biolabs) or KOD One (KMM-101, TOYOBO, Osaka, Japan) according to manufacturer’s instructions. The samples were incubated at 30°C for 45 min followed by a 15-min incubation at 42°C. After centrifugation and removal of supernatant, the cell pellets were resuspended with 50 μL of SC−Ura medium without carbon source (7.4 g/L Yeast nitrogen base without amino acids, #291940, Thermo Fisher Scientific; 855 mg/L CSM−Ura powder, DCS0161, FORMEDIUM, Hunstanton, UK; and 111 mg/L adenine sulfate, Nacalai tesque) and spread on a SCGal−Ura agar plate (20 g/L galactose, #075-00035, FUJIFILM Wako Chemicals; 6.7 g/L Yeast nitrogen base without amino acids, Thermo Fisher Scientific; 770 mg/L CSM−Ura powder, FORMEDIUM; 100 mg/L adenine sulfate, Nacalai tesque; and 20 g/L agar, #010-08725, FUJIFILM Wako Chemicals). The plates were incubated at 30°C for 4 days. The colonies were picked and streaked as patches on SCGal−Ura agar plates, and then incubated at 30°C for 1–2 days followed by colony PCR to check successful genome editing. Colony PCR was performed using Q5 DNA polymerase (New England Biolabs) or KOD One (TOYOBO) according to manufacturer’s instructions. The PCR-positive clones were cultured overnight in 2 mL of YPAD liquid medium. An aliquot (10 μL) of the overnight culture was spotted and streaked on a YPAD agar plate for single colony isolation (30°C for 2 days). Single colonies were picked and streaked on YPAD agar plate and SCDex−Ura agar plate (20 g/L glucose, #16806-25, Nacalai tesque; 6.7 g/L Yeast nitrogen base without amino acids, Thermo Fisher Scientific; 770 mg/L CSM−Ura powder, FORMEDIUM; 100 mg/L adenine sulfate, Nacalai tesque; and 20 g/L agar, #010-08725, FUJIFILM Wako Chemicals) to check the loss of the genome-editing plasmid. The Ura− clones were re-examined by colony PCR to be successfully genome-edited. The colony PCR-positive Ura− clones were used in the subsequent experiments.

Plasmid extraction from yeast cells

Plasmids were extracted from yeast cells by Easy Yeast Plasmid Isolation Kit (#630467, Takara Bio) and transformed into E. coli competent cells (Champion DH5α high, SMOBIO Technology).

Fluorescence microscopy and image processing

Image acquisitions of yeast cells were performed on a microscope (Ti-E, Nikon, Tokyo, Japan) with a 100× objective lens (CFI Apo TIRF 100XC Oil, MRD01991, Nikon), a sCMOS camera (ORCA-Fusion BT, C15440-20UP, Hamamatsu photonics, Hamamatsu, Japan), and a solid-state illumination light source (SOLA SE II, Lumencor, Beaverton, OR, USA). Image acquisition was controlled by NIS-Elements version 5.3 (Nikon). The binning mode of the camera was set at 2×2 (0.13 μm/pixel). Z-stacks were 13×0.3 μm. For imaging of Cse4-mNeonGreen, a filter set (LED-YFP-A, Semrock, Rochester, NY, USA) was used with excitation light power set at 20% and exposure time set at 200 msec/frame. For imaging of Cdc3-mScarlet-I, a filter set (LED-TRITC-A, Semrock) was used with excitation light power set at 7% and exposure time set at 70 msec/frame. For DIC (differential interference contrast) image acquisition, exposure time was set at 20 msec/frame. DIC images were captured only at the middle position of the Z-stacks.

Image processing and analysis were performed using Fiji (Schindelin et al. 2012). To generate 2-dimensional images of fluorescence channel from Z-stacks, background subtraction (sliding paraboloid radius set at 10 pixels with disabled smoothing) and maximum projection using 13 Z-slices were performed. Maximum projected fluorescence images and corresponding smoothed DIC images were superimposed. After global adjusting of brightness and contrast and cropping of the images, sequences of representative images were generated.

Editable fraction of yeast genome with three CRISPR/Cas systems

S. cerevisiae reference genome sequence available at Saccharomyces genome database (SGD) (S288C strain, version R64-2-1, http://sgd-archive.yeastgenome.org/sequence/S288C_reference/genome_releases/S288C_reference_genome_R64-2-1_20150113.tgz) without mitochondrial genome and plasmid sequences were searched for PAMs (NGG for SpCas9, NNGRRT for SaCas9, and TTTV for AsCas12a). Both strands were included in the PAM search. After identification of the PAM sequence, nucleotides in a defined distance from the PAM were assigned as candidate nucleotides for editing. In genome editing, it is critical for a successfully-edited target sequence in the genome not to be cleaved again by the Cas–gRNA complex bearing the gRNA corresponding to the original, unedited target sequence. We thus defined a nucleotide as a candidate for editing if its substitutions leading to mismatches with the gRNA can significantly reduce the efficiency of re-cleavage by the Cas–gRNA complex. For SpCas9 and SaCas9, the nucleotides at one to eleven nt away from the PAM and the nucleotides consisting the PAM were defined as the candidates based on previous reports (Anderson et al. 2015; Zheng et al. 2017; Tycko et al. 2018) (Figures S1A and S1B). For AsCas12a, the nucleotides that are one to seventeen nt away from the PAM and the nucleotides consisting the PAM were defined as the candidates (Kim et al. 2016; Kleinstiver et al. 2016; Bin Moon et al. 2018) (Figure S1C). Degenerate nucleotides in the PAMs (i.e., N, R, and V) were excluded from the calculation (Figures S1A, S1B, and S1C). The total number of the candidate nucleotides for editing is summarized in Table S5 and Figure S1D.

ORFs editable at their 5′ ends

S. cerevisiae ORF sequence collection available at SGD (http://sgd-archive.yeastgenome.org/sequence/S288C_reference/orf_dna/orf_genomic_all.fasta.gz) was used for the search of ORFs that can be edited at their 5′ ends. ORFs on the mitochondrial genome and ORFs on the two-micron plasmids were omitted from the analysis. The total number of ORFs analyzed in this study was 6,881. Candidate nucleotides for editing were searched in each sequence (ORF and upstream 1,000 and downstream 1,000 nt) by the method described above. When at least one nucleotide within the start codon ‘ATG’ was assigned as the candidate for editing, the ORF was categorized as an ORF that is editable at its 5′ end. The total number of the ORFs editable at the 5′ ends is summarized in Table S6 and Figure S1E.

Data availability

The four backbone vectors are available from NBRP Yeast Resource Center (https://yeast.nig.ac.jp/yeast/). The source codes of programs for ODN design, PAM search, and 5′-editable ORF search are available from our repository at GitHub (https://github.com/poccopen/Genome_editing_plasmid_for_budding_yeast). Other strains and plasmids are available upon request. The authors state that all data necessary for confirming the conclusions presented here are represented fully within the article.

Article TitleSimple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

Abstract

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.


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