Rapid assembly of gRNA arrays via modular cloning in yeast
File(s)
Author(s)
McCarty, Nicholas S
Shaw, William M
Ellis, Tom
Ledesma-Amaro, Rodrigo
Type
Journal Article
Abstract
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.
Date Issued
2019-04-02
Date Acceptance
2019-04-01
Citation
ACS Synthetic Biology, 2019, 8 (4), pp.906-910
ISSN
2161-5063
Publisher
American Chemical Society
Start Page
906
End Page
910
Journal / Book Title
ACS Synthetic Biology
Volume
8
Issue
4
Copyright Statement
© 2019 American Chemical Society
Sponsor
Engineering & Physical Science Research Council (EPSRC)
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/30939239
Grant Number
EP/M002306/1
Subjects
Science & Technology
Life Sciences & Biomedicine
Biochemical Research Methods
Biochemistry & Molecular Biology
yeast
MoClo toolkit
CRISPR
multiplex
assembly method
Golden Gate assembly
CRISPR
Golden Gate assembly
MoClo toolkit
assembly method
multiplex
yeast
Publication Status
Published online
Coverage Spatial
United States
Date Publish Online
2019-04-02