Expanded CRISPEY Kit
(Kit #
1000000257
)
Depositing Lab: Jeffrey Lewis
The Expanded CRISPEY Kit contains a suite of plasmids for creating genomic edits in Saccharomyces cerevisiae using CRISPR/Cas9 cleavage coupled with directed homologous recombination using an in vivo expressed repair template. A variety of mutations can be created using this system, including deletions, point mutations, and insertions. This kit can be used in laboratory, wild, and industrial strains that are haploid or diploid, with editing efficiencies ranging from 50% to >95%.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Stuecker TN, Hood SE, Pineda JM, Lenaduwe S, Winter J, Sadhu MJ, Lewis JA. bioRxiv. 2024 Aug 7:2024.08.06.606807. doi: 10.1101/2024.08.06.606807. PubMed (Link opens in a new window) Article (Link opens in a new window).
Description
The Expanded CRISPEY Kit contains six pCRISPEY plasmids, seven pCas9-EcRT plasmids, and five control plasmids. Each pCRISPEY plasmid contains a URA3 gene for auxotrophic selection and one of three antibiotic selection markers (Kanamycin, Hygromycin, or Nourseothricin). Each pCas9-EcRT plasmid contains one of four selectable markers (Kanamycin, Hygromycin, Nourseothricin, or HIS3). Two versions of each pCRISPEY and pCas9 plasmid are available with different induction systems (galactose-inducible or estradiol-inducible). The pCRISPEY control plasmids target the ADE2 gene.
The pCRISPEY plasmids contain a unique cloning site: NotI for galactose-inducible plasmids or XhoI for estradiol-inducible plasmids. A user-designed guide RNA (gRNA) and repair template (obtained separately) can be synthesized as a single oligo and cloned into the pCRISPEY vector. This cloning strategy typically has >95% cloning efficiency. The pCRISPEY plasmid is then co-transformed into yeast with a pCas9-EcRT plasmid. Upon induction, the gRNA and repair template are expressed from the pCRISPEY vector while the Ec86 reverse transcriptase and SpCas9 are expressed from pCas9-EcRT. The Ec86 reverse transcriptase amplifies the gRNA and repair template in vivo to increase the available template for homologous recombination, yielding high editing efficiencies (50–99%). Plasmids can be 'cured' from final strains to generate markerless genome edits, which would also enable making multiple edits sequentially.
Figure 1: The CRISPEY system pairs a pCRISPEY plasmid with a pCas9-EcRT plasmid for directed editing of the yeast genome. User-designed gRNA and homologous repair template are cloned into pCRISPEY at a unique NotI (for galactose-induced plasmids) or XhoI (for estradiol-induced plasmids) site.
Kit Documentation
CRISPEY-kit-manual (PDF, 2.3 MB)How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The Expanded CRISPEY Kit was a gift from Jeffrey Lewis (Addgene kit #1000000257).”
For your Reference section:
Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Stuecker TN, Hood SE, Pineda JM, Lenaduwe S, Winter J, Sadhu MJ, Lewis JA. bioRxiv. 2024 Aug 7:2024.08.06.606807. doi: 10.1101/2024.08.06.606807. PubMed (Link opens in a new window) Article (Link opens in a new window).
The Expanded CRISPEY Kit - #1000000257
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Ampicillin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pCas9-EcRT-GAL-Nat | Ampicillin |
| A / 2 | pCas9-EcRT-GAL-Hyg | Ampicillin |
| A / 3 | pCas9-EcRT-GAL-Kan | Ampicillin |
| A / 4 | pCas9-EcRT-GAL-HIS3 | Ampicillin |
| A / 5 | pCas9-EcRT-Z3-Nat | Ampicillin |
| A / 6 | pCas9-EcRT-Z3-Hyg | Ampicillin |
| A / 7 | pCas9-EcRT-Z3-Kan | Ampicillin |
| A / 8 | pCRISPEY-GAL-Nat | Ampicillin |
| A / 9 | pCRISPEY-GAL-Hyg | Ampicillin |
| A / 10 | pCRISPEY-GAL-Kan | Ampicillin |
| A / 11 | pCRISPEY-Z3-Nat | Ampicillin |
| A / 12 | pCRISPEY-Z3-Hyg | Ampicillin |
| B / 1 | pCRISPEY-Z3-Kan | Ampicillin |
| B / 2 | pCRISPEY-GAL-Nat-ADE2 | Ampicillin |
| B / 3 | pCRISPEY-GAL-Hyg-ADE2 | Ampicillin |
| B / 4 | pCRISPEY-GAL-Kan-ADE2 | Ampicillin |
| B / 5 | pCRISPEY-Z3-Nat-ADE2 | Ampicillin |
| B / 6 | pCRISPEY-Z3-Hyg-ADE2 | Ampicillin |