Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 Nov 22;8:17277. doi: 10.1038/s41598-018-34909-3

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Design of a CRISPR/Cas9-based gene drive system in S. cerevisiae across three loci. (A) Left, An artificial gene drive was installed at three loci in haploid yeast. Each drive system (Drive 1–3) contained a guide RNA cassette targeting an artificial target (Target 1–3) at the same locus. Only Drive 1 contained the cassette for S. pyogenes Cas9. Right, Artificial (u1) and (u2) sites⁶³ were used flanking the gene drive at the HIS3 locus (Chromosome XV) and the S.p.HIS5 selectable marker. The SHS1 locus (Chromosome IV) included a C-terminal GFP and C.a.URA3. DNL4 (Chromosome XV) was deleted with the Kan^R cassette. All sgRNAs were targeted to non-native sequences. The sgRNA(u1) cassette was on a high-copy plasmid (LEU2 marker). S.p.Cas9 was under control of the inducible GAL1/10 promoter. (B) Haploid yeast harboring the triple drive (GFY-3675) were mated to the triple target strain (GFY-3596) to form diploids. Cas9 expression was induced by galactose (0 or 5 hr). Cultures were diluted to 100–500 cells per plate, grown for 2 days, and transferred to SD-LEU, SD-HIS, SD-URA, and G418 plates. (C) A time course of galactose activation using the [GFY-3675 x GFY-3596] diploid in triplicate. Error, SD. (D) Seven haploid strains (GFY-3206, 3593, 3264b, 3578, 3594, 3623, and 3596) were tested as in (B) against the triple drive strain (GFY-3675). (E) Each of the diploids from (D) were cultured for 5 hr and quantified for drive success. Error, SD. (F) Clonal isolates were obtained from diploids generated in (B) at either 0 hr (2 isolates) or 5 hr activation of Cas9 (14 isolates). All yeast were confirmed as diploids and assayed on each media type (below). Diagnostic PCRs were performed on genomic DNA to detect the presence (or absence) of each locus; oligonucleotide (Supplementary Table 5) positions can be found in (A) and the expected sizes are illustrated (right). Two isolates (13,14) were chosen for their incomplete growth profile (red asterisks). Images were cropped from separate portions of larger gels or from independent DNA gels and are separated by white lines. The unedited images can be found in Supplementary Fig. S8.