Broadening the Nicotiana benthamiana research toolbox through the generation of dicer-like mutants using CRISPR/Cas9 approaches

RNA silencing in plants plays a pivotal role in various biological processes, including development, epigenetic modifications and stress response. Key components of this network are Dicer-like (DCL) proteins. Nicotiana benthamiana encodes four DCLs, each responsible for the generation of distinct small RNA (sRNA) populations, which regulate different functions. However, elucidating the precise role of each DCL has been proven challenging, as overlapping functions exist within DCLs. In our present study, we have successfully generated dcl2, dcl3 and dcl4 homozygous mutants, employing two different CRISPR/Cas9 approaches. The first approach is based on a transgene-mediated delivery of the single-guide RNA (sgRNA), while the second approach employs a viral vector for sgRNA delivery. By utilizing a suite of screening techniques, including polymerase chain reaction (PCR), T7 endonuclease I (T7E1) assay, high-resolution melt analysis (HRMA) and DNA sequencing, we successfully generated dcl2, dcl3 and dcl4 homozygous mutants harboring identical mutations in every allele. To evaluate these dcl mutants, we examined their sRNA profiles and phenotypes. We further have indications that homozygous mutations of a gene do not always lead to the desired loss-of-function, highlighting the importance of mutant evaluation. dcl mutants represent invaluable tools to explore how overlapping silencing pathways are connected to essential plant functions, including development, stress responses and pathogen defense. Additionally, they hold potential for biotechnological applications, such as crop improvement and gene silencing tools. We anticipate that our study will make significant contributions to enhance understanding of the role of DCLs in plants.