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ACS Synth Biol. 2019 Apr 19;8(4):906-910. doi: 10.1021/acssynbio.9b00041. Epub 2019 Apr 11.

Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast.

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Bioengineering , California Institute of Technology , Pasadena , California 91125 , United States.
Imperial College Centre for Synthetic Biology , Imperial College London , London , SW7 2AZ , U.K.
Department of Bioengineering , Imperial College London , London , SW7 2AZ , U.K.


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.


CRISPR; Golden Gate assembly; MoClo toolkit; assembly method; multiplex; yeast


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