Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae   

Materials and Reagents

1. Escherichia coli: DH5ɑ (sanyou Biopharmaceuticals)
   
2. S. cerevisiae strain: W303 (MATa ura3-52)
   
3. pCRCT plasmid (Addgene, catalog number: 60621 )
   
4. Salmon DNA (Sigma-Aldrich, catalog number: D1626 )
   
5. Restriction enzyme: 10 U/μl Bsal (Takara Bio)
   
6. 10x buffer G (Takara Bio)
   
7. Taq DNA polymerase (Takara Bio)
   
8. Antibiotic: 100 µg/ml ampicillin (Siyao)
   
9. Pure plasmid mini kit (CWBIO)
   
10. Yeast Gen DNA Kit (CWBIO)
    
11. DNA oligonucleotide primers (GENEWIZ)
    
12. PEG: Poly (ethylene glycol), BioXtra avg. molecular weight 3,350 (Sigma-Aldrich, catalog number: P3640 )
    Note: This product has been discontinued.
    
13. Yeast extract (Oxoid, catalog number: LP0021 )
    
14. Tryptone (Oxoid, catalog number: LP0042 )
    
15. Dextrose
    
16. Agar (Solarbio, catalog number: A8190 )
    
17. Sodium chloride (NaCl) (Tianjin Kemiou Chemical Reagent)
    
18. Peptone
    
19. Primers
    CasYZF: 5’---ACGCTGTAGAAGTGAAAGTTGG---3’
    CasYZR: 5’---TAGTATGCTGTGCTTGGGTGTT---3’
    
20. Glucose (Tianjin Kemiou Chemical Reagent)
    
21. Adenine (Sigma-Aldrich)
    
22. Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883-1KG )
    
23. Agarose gel recovery kit (Biomiga)
    
24. YPD liquid media (see Recipes)
    
25. LB plates (with appropriate antibiotics included) (see Recipes)
    
26. YPDA liquid medium (see Recipes)
    
27. SC medium without uracil (see Recipes)
    

Equipment

1. PCR machine (or similar) (Biometra, model: TPfofessional )
   
2. 42 °C water bath (or similar) (XINBAO, catalog number: HH-501BS )
   
3. DNA electrophoresis apparatus (or similar) (SIM International, model: BIO-PRO 200E , catalog number: 0401RHSI049)
   
4. Microcentrifuge (SCILOGEX, model: D2012 )
   
5. Incubator (or similar, capable of incubation of agar plates at 37 °C or 28 °C) (CIMO, model: DNP-III )
   

Procedure

1. Find non-essential S. cerevisiae genes (after deletion or inactivation of which will not result in lethality) with one of the following databases:
   http://www.yeastgenome.org/
   http://mips.gsf.de/genre/proj/yeast/
   http://www.ncbi.nlm.nih.gov/
   
   
2. Design two 20 nucleotide (nt) spacer sequences for the CRISPR array
   Design the two 20 nt spacer sequences for the CRISPR array with the online tool E-CRISP. The following factors should be considered when designing the spacer sequence:
   
   1. Spacer sequence length is generally 17-20 nt. The protospacer adjacent motif (PAM) for the Streptococcus pyogenes Cas9 (SpCas9) is 5’-NGG-3’.
      
   2. The spacer sequence may be located on the non-transcribed strand or the transcribed strand.
      
   3. The selected spacer sequence should be specific to avoid off-target effects.
      The tool E-CRISP helps design site-specific spacer sequences.
      (http://www.e-crisp.org/E-CRISP/)
      
      
3. Construct the crRNA-Cas9 plasmid
   
   1. The dsDNA insertion fragment for the crRNA transcript which contains two spacer sequences and two partial direct repeat sequences has to be synthesized (Figure 1).
      
      
      Figure 1. Schematic representation of the dsDNA used for crRNAs expression
      
      
   2. The dsDNA insert for crRNA transcription is digested using BsaI and cloned into the BsaI-digested pCRCT plasmid (Bao et al., 2015).
   1. DNA digestion reaction:
      30 ng of pCRCT plasmid DNA (or the dsDNA)
      10x buffer G:                                                                2 µl
      Bsal:                                                                             1 µl
      Add H₂O to                                                                   20 µl
      
   2. Incubate at 37 °C for 3 h.
      
   3. Analyze the digestion reaction with 1% (or 2%) (w/v) agarose gel electrophoresis and recover the digested DNA using an agarose gel purification kit.
      
   3. Ligate the dsDNA insert for crRNA transcription into pCRCT plasmids
   1. The ligation reaction:
      BsaI digested plasmid:                                                     2 µl (20 ng)
      BsaI digested dsDNA insert for crRNA transcription:       0.3 µl (20-50 ng)
      Ligation mixture:                                                               2.5 µl
      Add H₂O to                                                                       5 µl
      
   2. Incubate at 16 °C overnight.
      
   4. Transform E. coli DH5α competent cells with the ligation product.
   1. Add 5 μl ligation mix into ice-cold competent E. coli DH5α (100 μl).
      
   2. Incubate mixture on ice for 30 min and heat-shock it at 42 °C for 90 sec.
      
   3. Cool down on ice for 2 min.
      
   4. Add 700 μl LB medium (without antibiotics) and incubate at 37 °C and shake at 250 rpm for 45 min.
      
   5. Plate 200 μl bacteria suspension on LB plate containing 100 μg/ml ampicillin.
      
   6. Incubate overnight at 37 °C in an incubator.
      
   7. Run a colony PCR for colonies per transformed pCRCT with CasYZF/CasYZR primers.
      PCR protocol
      95 °C     5 min       1 cycle
      95 °C     30 sec
      55 °C     30 sec     30 cycles
      72 °C     1 min
      72 °C     5 min       1 cycle
      4 °C       Hold         1 cycle
      
   8. Verify the product by sequencing.
      
   9. Pick positive clone(s) in 5 ml LB medium with 100 µg/ml ampicillin and incubate for 16 h, at 37 °C, 200 rpm in a shaker.
      
   5. Extract plasmids from the transformed DH5α cells using the Pure Plasmid Mini Kit.
      
   6. Transform crRNA-Cas9 plasmids into S. cerevisiae competent cells using LiAc/SS carrier DNA/PEG method (Gietz et al., 2007).
      
      
4. Verification of Cas9-crRNA mediated large fragment deletions in S. cerevisiae.
   
   1. To verify Cas9-crRNA mediated large fragment deletions, a pair of gene specific primers is required to amplify the targeted region.
      
   2. Pick positive clone(s) from selection plate in 5 ml SC medium without uracil and incubate at 28 °C and shake at 200 rpm for 4 d.
      
   3. Extract S. cerevisiae genomic DNA from step D2 using Yeast Gen DNA Kit.
      
   4. Perform PCR amplification using genomic DNAs extracted from transgenic S. cerevisiae with the following PCR procedure:
      Note: P₁ and P₂ primers (Figure 2, Table 1) are designed based on the sequence flanking the target region to amplify the junction sequence, and amplicon is possible across the ligated junction only when large deletion occurred. P₃-P₈ primers can confirm the result. The primers can be designed by Primer3 (length: 18-25, GC content: 40%-60%).
      PCR protocol
      95 °C     5 min     1 cycle
      95 °C     30 sec
      X °C       30 sec   30 cycles
      72 °C     N min
      72 °C     5 min     1 cycle
      4 °C       Hold      1 cycle
      Note: Annealing temperature (X) and extension time (N) depend on primers.
      
      
      
      Figure 2. Large fragment deletion by CRISPR/Cas9 as described in this protocol.
      
      See Table 1 for the sequences of P₁-P₈, gRNA1 and gRNA2
      
      
      Table 1. The sequences of P1-P8, gRNA1 and gRNA2
      
      
      
      
   5. The PCR product is verified by electrophoresis (Figure 3).
      
      
      Figure 3. Confirmation of the large chromosomal modifications. A. M, marker; lane 1, mutant; lane 2, control. B, C and D. The PCR product with primers P₃-P₄, P₅-P₆, P₇-P₈ in the target region, respectively, 1a, 1b, 1c, the parental strain. 2a, 2b, 2c, the deletant. 
      

Notes

In this protocol, the large-scale deletion efficiency was only 10%. The likely reason for this is that the DSB can be repaired via NHEJ, which typically results in small insertions and/or deletions (indels) at the site of the break. While the HDR pathway allows high fidelity and precise editing, the efficiency of large-scale deletion in S. cerevisiae could be improved if the target vector contains the homology arms of target gene.

Recipes

Note: The solvent for the media is ddH₂O.

1. YPD liquid media
   1% (w/v) yeast extract
   2% (w/v) tryptone
   2% (w/v) dextrose
   Autoclave and store at 4 °C
   
2. LB plates (with appropriate antibiotics included)
   1.5% (w/v) agar
   1% (w/v) tryptone
   0.5% (w/v) yeast extract
   1% (w/v) NaCl
   Autoclave
   
3. YPDA liquid medium
   2% (w/v) peptone
   2% (w/v) glucose
   1% (w/v) yeast extract
   0.005% (w/v) adenine
   Autoclave and store at 4 °C
   
4. SC medium without uracil
   2% (w/v) glucose
   0.67% (w/v) yeast nitrogen base
   Autoclave and store at 4 °C
   

Acknowledgments

This work was supported by the open fund of Key laboratory (No. 3333112) and Bioengineering key discipline of Hebei Province.

References

1. Bao, Z., Xiao, H., Liang, J., Zhang, L., Xiong, X., Sun, N., Si, T. and Zhao, H. (2015). Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol 4(5): 585-594.
   
2. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. and Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819-823.
   
3. Gietz, R. D. and Schiestl, R. H. (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 1-4.
   
4. Hirashima, K., Iwaki, T., Takegawa, K., Gigahama, Y. and Tohda, H. (2006). A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast. Nucleic Acids Res 34(2): e11.
   
5. Varshney, G. K., Pei, W., LaFave, M. C., Idol, J., Xu, L., Gallardo, V., Carrington, B., Bishop, K., Jones, M., Li, M., Harper, U., Huang, S. C., Prakash, A., Chen, W., Sood, R., Ledin, J. and Burgess, S. M. (2015). High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Res 25(7): 1030-1042.
   
6. Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H. and Yang, B. (2014). Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42(17): 10903-10914.