Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast
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.
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.Attached Files
Supplemental Material - sb9b00041_si_001.pdf
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Additional details
- Eprint ID
- 94391
- Resolver ID
- CaltechAUTHORS:20190403-081411236
- BB/R01602X/1
- Biotechnology and Biological Sciences Research Council (BBSRC)
- BB/K019791/1
- Biotechnology and Biological Sciences Research Council (BBSRC)
- Fulbright Foundation
- Imperial College London
- Created
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2019-04-03Created from EPrint's datestamp field
- Updated
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2021-11-16Created from EPrint's last_modified field