CRISPR-Cas12a nucleases function with structurally engineered crRNAs: SynThetic trAcrRNA

CRISPR-Cas12a systems are becoming an attractive genome editing tool for cell engineering due to their broader editing capabilities compared to CRISPR-Cas9 counterparts. As opposed to Cas9, the Cas12a endonucleases are characterized by a lack of trans-activating crRNA (tracrRNA), which reduces the complexity of the editing system and simultaneously makes CRISPR RNA (crRNA) engineering a promising approach toward further improving and modulating editing activity of the CRISPR-Cas12a systems. Here, we design and validate sixteen types of structurally engineered Cas12a crRNAs targeting various immunologically relevant loci in-vitro and in-cellulo. We show that all our structural modifications in the loop region, ranging from engineered breaks (STAR-crRNAs) to large gaps (Gap-crRNAs), as well as nucleotide substitutions, enable gene-cutting in the presence of various Cas12a nucleases. Moreover, we observe similar insertion rates of short HDR templates using the engineered crRNAs compared to the wild-type crRNAs, further demonstrating that the introduced modifications in the loop region led to comparable genome editing efficiencies. In conclusion, we show that Cas12a nucleases can broadly utilize structurally engineered crRNAs with breaks or gaps in the otherwise highly-conserved loop region, which could further facilitate a wide range of genome editing applications

Keep calm and carry on: miRNA biogenesis under stress

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression. Their biogenesis relies on the cleavage of longer precursors by a nuclear localized processing machinery. The evolutionary preference of plant miRNAs to silence transcription factors turned these small molecules into key actors during growth and adaptive responses. Furthermore, during their life cycle plants are subject to changes in the environmental conditions surrounding them. In order to face these changes, plants display unique adaptive capacities based on an enormous developmental plasticity, where miRNAs play central roles. Many individual miRNAs have been shown to modulate the plant response to different environmental cues and stresses. In the last few years, increasing evidence has shown that not only individual genes encoding miRNAs but also the miRNA pathway as a whole is subject to regulation in response to external stimulus. In this review, we discuss the current knowledge about the miRNA pathway. We dissect the pathway to analyze the events leading to the generation of these small RNAs and emphasize the regulation of core components of the miRNA biogenesis machinery.Fil: Manavella, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Yang, Seong W.. Yonsei University; Corea del SurFil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin