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Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast

McCarty, Nicholas S. and Shaw, William M. and Ellis, Tom and Ledesma-Amaro, Rodrigo (2019) Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast. ACS Synthetic Biology, 8 (4). pp. 906-910. ISSN 2161-5063.

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CRISPR is a versatile technology for genomic editing and regulation, but the expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and expressed from a single transcriptional unit. We demonstrate that these gRNA arrays can stably regulate gene expression in a synergistic manner via dCas9-mediated repression. This approach expands the number of gRNAs that can be expressed in this model organism and may enable the versatile editing or transcriptional regulation of a greater number of genes in vivo.

Item Type:Article
Related URLs:
URLURL TypeDescription Information
Ellis, Tom0000-0001-5392-976X
Ledesma-Amaro, Rodrigo0000-0003-2631-5898
Additional Information:© 2019 American Chemical Society. Received: February 2, 2019; Published: April 2, 2019. This work was supported by BBSRC Grant (BB/R01602X/1) (to R.L.A.), BBSRC Grant BB/K019791/1 (to T.E.) and a Fulbright–Imperial College London Partnership award (to N.S.M.). The authors declare the following competing financial interest(s): A patent application related to this work has been filed by the authors.
Funding AgencyGrant Number
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R01602X/1
Biotechnology and Biological Sciences Research Council (BBSRC)BB/K019791/1
Fulbright FoundationUNSPECIFIED
Imperial College LondonUNSPECIFIED
Subject Keywords:yeast, MoClo toolkit, CRISPR, multiplex, assembly method, Golden Gate assembly
Issue or Number:4
Record Number:CaltechAUTHORS:20190403-081411236
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Official Citation:Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast. Nicholas S. McCarty, William M. Shaw, Tom Ellis, and Rodrigo Ledesma-Amaro. ACS Synthetic Biology 2019 8 (4), 906-910. DOI: 10.1021/acssynbio.9b00041
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:94391
Deposited By: Tony Diaz
Deposited On:03 Apr 2019 18:23
Last Modified:03 Oct 2019 21:03

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