TY - JOUR
T1 - A Suite of New Strain Construction Vectors for Gene Expression Knockdown in Budding Yeast
AU - Shively, Christian A.
AU - Dong, Fengping
AU - Mitra, Robi D.
N1 - Funding Information:
This work was supported by RF1MH117070, RF1MH126723 (National Institute of Mental Health) (R.D.M.), and R01GM123203 (National Institute of General Medical Sciences) (R.D.M.).
Funding Information:
We wish to thank Anton Khmelinskii for the C-SWAT collection and associated plasmids. Linda Riles provided invaluable expertise in replicating and storing the C-SWAT collection. Kyle Cunningham provided plasmid pAIDA2(6FLAG). Tamara Abou-Antoun provided useful feedback and instruction in immunoblotting. EUROSCARF (www.euroscarf.de) provided plasmid pMK632 and yeast strain YMK120. This work was supported by RF1MH117070, RF1MH126723 (National Institute of Mental Health) (R.D.M.), and R01GM123203 (National Institute of General Medical Sciences) (R.D.M.).
Publisher Copyright:
© 2023 American Chemical Society.
PY - 2023/2/17
Y1 - 2023/2/17
N2 - Numerous tools for gene expression knockdown have been developed and characterized in the model organism Saccharomyces cerevisiae and extended to facilitate studies in multicellular models. To comparatively evaluate the efficacy of these approaches, we systematically applied seven such published constitutive and inducible knockdown strategies to a panel of essential genes encoding nuclear-localized proteins. In this effort, we created the CEAS (C-SWAT for Essential Allele Strains) collection, a suite of tagging vectors for improved utility and ease of strain construction. Of particular note, we adapted an improved auxin inducible degron (AID) protein degradation strategy previously available only in mammalian tissue culture for one-step strain construction in budding yeast by leveraging both the C-SWAT system and CRISPR/Cas9 editing. Taken together, this work presents a toolbox for endogenous gene expression knockdown and allows us to make recommendations on the efficacy and applicability of these tools for the perturbation of essential genes.
AB - Numerous tools for gene expression knockdown have been developed and characterized in the model organism Saccharomyces cerevisiae and extended to facilitate studies in multicellular models. To comparatively evaluate the efficacy of these approaches, we systematically applied seven such published constitutive and inducible knockdown strategies to a panel of essential genes encoding nuclear-localized proteins. In this effort, we created the CEAS (C-SWAT for Essential Allele Strains) collection, a suite of tagging vectors for improved utility and ease of strain construction. Of particular note, we adapted an improved auxin inducible degron (AID) protein degradation strategy previously available only in mammalian tissue culture for one-step strain construction in budding yeast by leveraging both the C-SWAT system and CRISPR/Cas9 editing. Taken together, this work presents a toolbox for endogenous gene expression knockdown and allows us to make recommendations on the efficacy and applicability of these tools for the perturbation of essential genes.
UR - http://www.scopus.com/inward/record.url?scp=85146618790&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.2c00547
DO - 10.1021/acssynbio.2c00547
M3 - Article
C2 - 36650116
AN - SCOPUS:85146618790
SN - 2161-5063
VL - 12
SP - 624
EP - 633
JO - ACS synthetic biology
JF - ACS synthetic biology
IS - 2
ER -