Positional effects on efficiency of CRISPR/Cas9-based transcriptional activation in rice plants

The nuclease-dead Cas9 (dCas9) has been reprogrammed for transcriptional activation by fusing dCas9 to a transcriptional activation domain. In the presence of a guide RNA (gRNA), the dCas9 fusions specifically bind to regions of a promoter to activate transcription. Significant amount of effort has been directed toward the identification and optimization of the fusions of dCas9-activation domain, but very little is known about the impact of gRNA target positions within a promoter in plants on transcriptional activation efficiency. The dCas9–6TAL–VP128 system (dCas9-TV) has been optimized to activate transcription in plants. Here we use the dCas9-TV to activate transcription of OsWOX11 and OsYUC1 , two genes that cause dramatic developmental phenotypes when overexpressed. We designed a series of gRNAs targeting the promoters of the two genes. We show that gRNAs that target regions within 350 bp upstream of the transcription start site were most effective in transcriptional activation. Moreover, we show that using two gRNAs that simultaneously target two discrete sites in a promoter can further enhance transcription. This work provides guidelines for designed transcriptional activation through CRISPR/dCas9 systems.