2026-09-15T04:10:45Zhttps://gredos.usal.es/oai/request

oai:gredos.usal.es:10366/1507442025-04-30T20:40:53Zcom_10366_149960com_10366_4512com_10366_3823col_10366_149961

00925njm 22002777a 4500 dc Jiménez García, Alberto author Hoff, Birgit author Revuelta Doval, José Luis author 2020-03 [EN]CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained by the existence of a specific5′-NGG-3′ PAM sequence in the target site. Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows a T-rich PAM sequence (5′-TTTN-3′)to be employed and facilitates implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions with five different auxotrophic markers (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system is demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multiCRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may affect significantly the editing efficiency of the system. http://hdl.handle.net/10366/150744 10.1016/j.nbt.2020.02.002 Ashbya gossypii CRISPR/Cpf1 Genome engineering multiplex gene editing Filamentous fungi Ashbya gossypii CRISPR/Cpf1 Genome engineering Multiplex gene editing Filamentous fungi Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1