Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit
(Kit #
1000000210
)
Depositing Lab: Vincent Martin
The aim of the MyLO CRISPR-Cas9 toolkit is to improve efficiency, and decrease complexity, of common markerless manipulations of the yeast genome. This is achieved with a suite of vectors with Cas9 and either complete guides or guide cloning sites with GFP dropout cassettes, which facilitate transformation-based cloning or Golden Gate cloning. The Golden Gate cloning Cas9 vectors contain novel split selection cassettes that improve transformation efficiencies. The kit also includes parent vectors for Golden Gate cloning target genes into integration cassettes with either N- or C-terminal GFP, RFP, or 3xHA tags. Finally, a suite of integration cassettes featuring validated GFP- or RFP-tagged compartmental markers is included.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit. Bean BDM, Whiteway M, Martin VJJ. G3 (Bethesda). 2022 Jul 29;12(8):jkac154. doi: 10.1093/g3journal/jkac154. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
This toolkit contains Cas9-gRNA vectors, integration plasmids, and organellar localization markers as a versatile set of plasmids for Cas9-mediated integration of tagged genes and markers into the yeast genome. The Cas9-gRNA vectors contain one of five selectable markers (KanR, HygR, NatR, LEU2, URA3). gRNAs are introduced through transformation-based cloning into vectors with full selection markers, Golden Gate cloning into vectors with novel split selection markers that improve transformation efficiencies, or using pre-cloned gRNA targeting seven common sites. A set of Golden Gate cloning-compatible plasmids allow quick production of integration cassettes for a gene of interest (GOI) with three different N- or C-terminal tags (GFP, RFP, HA) at three expression levels. These cassettes have multi-target homology arms targeting seven different safe harbor sites. This toolkit also includes a validated set of 21 integration-ready GFP- or RFP-tagged organellar localization markers for use in colocalization studies.
Kit-Specific Resources
Toolkit details, instructions, and recommended protocols are available in Supplemental File S2 of the original publication (PDF, 3.4 MB) (Link opens in a new window)
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 Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit was a gift from Vincent Martin (Addgene kit #1000000210).”
For your Reference section:
The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit. Bean BDM, Whiteway M, Martin VJJ. G3 (Bethesda). 2022 Jul 29;12(8):jkac154. doi: 10.1093/g3journal/jkac154. PubMed (Link opens in a new window) Article (Link opens in a new window)
The Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit - #1000000210
- 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 | |
| Kanamycin | |
| Hygromycin |