CRISPR-Cas9 knock-in at the ChrXIII Int8 locus

Adapted from the lab compendium Snowflake Yeast Protocols — Ratcliff Lab / MuLTEE (V2).

CRISPR-based knock-in transformation (constitutive or inducible donor)

This system enables single-locus gene knock-ins (e.g., GFP labeling) into the Y55 background using Golden Gate assembly (GGA) and CRISPR-Cas9-mediated homologous recombination, then delivered via the standard LiAc/PEG transformation protocol (Transformation › Lithium acetate/PEG transformation of snowflake yeast). Adapted from a Multiplex MoClo Toolkit for engineering S. cerevisiae, which describes ten candidate genomic integration loci (Shaw, Khalil & Ellis, ACS Synthetic Biology 12:3393–3405, 2023); of these, only one — Int8 on Chromosome XIII — has a Y55-assembled reference sequence deposited in SGD, and it is the locus currently used in this lab (internal protocol, v. 2026-04-17).

Overview. Two plasmids are required, both flanked by NotI sites so the functional cassette can be excised before transformation:

1. CRISPR-Cas9 plasmid (targets the ChrXIII Int8 locus): NotI–Cas9–gRNA–gRNA scaffold–NotI.
2. Donor plasmid (carries the insertion cassette and homology arms): NotI–upstream homology arm–promoter–insertion sequence–terminator/selection marker cassette (hygromycin resistance)–downstream homology arm–NotI.
   • Constitutive donor: driven by the TEF promoter.
   • Inducible donor: promoter replaced with a synthetic inducible promoter.

Plasmid construction (Golden Gate assembly).

1. Order any needed synthesized DNA fragments with the appropriate restriction sites added at both ends. The insertion sequence itself must not contain internal BbsI sites.
2. PCR-amplify the remaining fragments (gRNA, homology arms, promoter, insertion sequence, marker cassette) from the templates specified in the lab’s plasmid records, using the corresponding primer pairs.
3. Assemble each plasmid by Golden Gate assembly: the CRISPR-Cas9 plasmid via BsaI-based GGA (backbone + gRNA insert); the donor plasmid via BbsI-based GGA (backbone + homology arms + promoter + insertion + marker cassette).
4. Reaction mix (10 μL total): 0.5 μL backbone (25 nM), 0.5 μL per insert (50 nM), 1 μL 10× T4 DNA Ligase Buffer, 0.5 μL Golden Gate enzyme mix (or 0.5 μL BsaI/BbsI plus 0.5 μL T4 DNA ligase — double both if assembling ≥10 inserts), water to 10 μL.
5. Thermocycle as repeated digestion–ligation cycles (digestion 2 min at 37 °C for BsaI/BpiI or 42 °C for BsmBI; ligation 5 min at 16 °C), roughly 10 cycles per insert (10 cycles for 1 insert, up to 25+ cycles for 4 or more inserts), followed by a final digestion (55 °C, 10 min) and heat inactivation (80 °C, 10 min).
6. Verify the completed donor plasmid by full-plasmid sequencing before use.

Transformation.

1. Digest both the CRISPR-Cas9 plasmid and the donor plasmid with NotI to release the functional cassettes from their backbones.
2. Transform the two digested fragments together into snowflake yeast cells using the standard LiAc/PEG transformation protocol (Transformation › Lithium acetate/PEG transformation of snowflake yeast).
3. Select transformants on hygromycin (resistance marker carried on the donor cassette).

Changing the insertion. Only the donor plasmid needs to be rebuilt to target a different insertion at the same ChrXIII locus: repeat the Golden Gate assembly with a donor insert amplified using a new primer set, and reuse the same CRISPR-Cas9 plasmid, which is locus-specific rather than insertion-specific.

⚠️ Forward-primer adapter sequences for the insertion fragment differ between constitutive and inducible donor constructs. Consult current lab plasmid records for the correct BbsI adapter sequences before ordering primers — these are lab/construct-specific and easy to get wrong.