McCarty Nicholas S, Shaw William M, Ellis Tom, Ledesma-Amaro Rodrigo
Bioengineering , California Institute of Technology , Pasadena , California 91125 , United States.
Imperial College Centre for Synthetic Biology , Imperial College London , London , SW7 2AZ , U.K.
ACS Synth Biol. 2019 Apr 19;8(4):906-910. doi: 10.1021/acssynbio.9b00041. Epub 2019 Apr 11.
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是一种用于基因组编辑和调控的通用技术，但到目前为止，酿酒酵母中多个gRNA的表达一直受到限制。我们在此展示了对酵母模块化克隆工具包的一个简单扩展，它能够使用最少的一组元件快速组装gRNA阵列。采用双PCR、IIs型限制酶的金门组装方法，至少12个gRNA可以从单个转录单元组装并表达。我们证明，这些gRNA阵列可以通过dCas9介导的抑制以协同方式稳定调控基因表达。这种方法增加了可在这种模式生物中表达的gRNA数量，并可能在体内实现对更多基因的通用编辑或转录调控。
