The evaluation of active transcriptional repressor domain for CRISPRi in plants

With the development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system in gene editing, the catalytic site in Cas9 has been mutated to dead Cas9 (dCas9) to regulate target gene's expression with the guidance of single guide RNA (sgRNA) in many organisms. When dCas9 was navigated to the region close to the transcription start site, duo to sterical hindrance, it could downregulate the expression level of target gene specifically without genomic alteration. Furthermore, the fusion of synthetic transcriptional repressor domain (TRD) to dCas9 could improve the gene silencing efficiency dramatically, the above all was also known as CRISPR interference system (CRISPRi). Till now, SRDX repressor domain was the most frequently used TRD in plant. Nevertheless, its incomplete repression limited the application of CRISPRi system. Hereafter, in this study, we identified three more effective TRDs, DLN144, DLS and MIX in plant. To dissect the transcriptional repressing activity of DLN144, DLS and MIX in plant, first and foremost, we proved their transcriptional repression efficiency in transient transformed Nicotiana benthamiana leaves. Then, their intrinsic transcriptional repressing activity was corroborated in stable transgenic wheat and N. benthamiana. These three functional TRDs, DLN144, DLS and MIX, provide more options for the application of CRISPRi in plant and shed new light on the advancement of more robust TRDs by combining different individual effective repressor domain in plant which will facilitate the application of CRISPRi when higher repression efficiency is required.