Home › Microbiology › CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art
Microbiology JoVE (Open Access) Citable · DOI

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

DOI: 10.3791/59350-v
What you'll learn
  • ✓ Design and express crRNA arrays for multiplex CRISPR/Cas12a genome editing
  • ✓ Transform S. cerevisiae with Cas12a and crRNA array constructs
  • ✓ Evaluate genome editing efficiency using carotenoid phenotype selection
  • ✓ Apply multiplex editing to create programmable cell-based visual displays
Protocol

Biopharma Insights The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S. cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

Difficulty
advanced
Total time
~5–7 days (including strain construction, transformations, and colony selection)
Model organism
Saccharomyces cerevisiae
Biosafety
BSL-1

Steps

1
Design single crRNA array expression cassette

Construct and prepare the single crRNA array expression cassette for multiplex targeting. This cassette enables simultaneous expression of multiple guide RNAs from a single transcript.

▶ 01:22
2
Perform first S. cerevisiae transformation

Transform S. cerevisiae with the Cas12a and crRNA array constructs using chemical or electroporation methods. Plate transformed cells and select on appropriate antibiotic resistance markers.

▶ 02:10
3
Perform second transformation of edited strains

Introduce additional editing constructs or selection markers into the first-generation transformants to complete multiplex genome modifications.

▶ 05:02
4
Evaluate editing efficiency and characterize phenotype

Assess multiplex genome editing success by analyzing carotenoid production phenotype in edited colonies, then arrange strains to create visual pixel art patterns.

▶ 06:42
5
Verify transformation and visualize yeast pixel art

Confirm successful genome editing through genotypic analysis and document the phenotypic pixel art created by the carotenoid-producing strain array.

▶ 08:30
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