RNA‐guided transcriptional regulation <i>in planta</i> via synthetic <scp>dC</scp>as9‐based transcription factors

<jats:title>Summary</jats:title><jats:p>Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR‐associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (<jats:styled-content style="fixed-case">dC</jats:styled-content>as9) protein combined with guide‐RNAs (<jats:styled-content style="fixed-case">gRNA</jats:styled-content>s) as a DNA‐targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA‐targeting platform for targeted transcriptional regulation <jats:italic>in planta</jats:italic> by developing chimeric <jats:styled-content style="fixed-case">dC</jats:styled-content>as9‐based transcriptional activators and repressors. To generate transcriptional activators, we fused the <jats:styled-content style="fixed-case">dC</jats:styled-content>as9 C‐terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the <jats:styled-content style="fixed-case">dC</jats:styled-content>as9 C‐terminus with the SRDX repression domain. Our data demonstrate that <jats:styled-content style="fixed-case">dC</jats:styled-content>as9 fusion with the EDLL activation domain (<jats:styled-content style="fixed-case">dC</jats:styled-content>as9:EDLL) and the TAL activation domain (<jats:styled-content style="fixed-case">dC</jats:styled-content>as9:TAD), guided by <jats:styled-content style="fixed-case">gRNA</jats:styled-content>s complementary to selected promoter elements, induce strong transcriptional activation on <jats:italic>Bs3::uidA</jats:italic> targets in plant cells. Further, the <jats:styled-content style="fixed-case">dC</jats:styled-content>as9:SRDX‐mediated transcriptional repression of an endogenous gene. Thus, our results suggest that the synthetic transcriptional repressor (<jats:styled-content style="fixed-case">dC</jats:styled-content>as9:SRDX) and activators (<jats:styled-content style="fixed-case">dC</jats:styled-content>as9:EDLL and <jats:styled-content style="fixed-case">dC</jats:styled-content>as9:TAD) can be used as endogenous transcription factors to repress or activate transcription of an endogenous genomic target. Our data indicate that the CRISPR/<jats:styled-content style="fixed-case">dC</jats:styled-content>as9 DNA‐targeting platform can be used in plants as a functional genomics tool and for biotechnological applications.</jats:p>