AMBIENTUM BIOETHICA BIOLOGIA CHEMIA DIGITALIA DRAMATICA EDUCATIO ARTIS GYMNAST. ENGINEERING EPHEMERIDES EUROPAEA GEOGRAPHIA GEOLOGIA HISTORIA HISTORIA ARTIUM INFORMATICA IURISPRUDENTIA MATHEMATICA MUSICA NEGOTIA OECONOMICA PHILOLOGIA PHILOSOPHIA PHYSICA POLITICA PSYCHOLOGIA-PAEDAGOGIA SOCIOLOGIA THEOLOGIA CATHOLICA THEOLOGIA CATHOLICA LATIN THEOLOGIA GR.-CATH. VARAD THEOLOGIA ORTHODOXA THEOLOGIA REF. TRANSYLVAN
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Authors: IUSTIN-TIBERIUS MUNTEANU, FAKHRI KALLABI, MARIUS MIHĂȘAN.
Published Online: 2021-06-30
Published Print: 2021-06-30
pp. 40-41
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ABSTRACT: Paenarthrobacter nicotinovorans is a Gram-positive bacterium that is best known for its ability to metabolize nicotine. The stain has proven its potential for converting nicotine containing waste into useful chemicals (Hritcu and Mihasan, 2019; Yu et al., 2017). Its applications in biotechnology are hampered by the lack of reliable gene editing systems that would permit rational engineering of the nicotine degradation pathway. CRISPR systems have been extensively used for genomic editing of eukaryotic cells and proved to be reliable and accurate. The applicability of CRISPR system for genomic editing of Paenarthrobacter strains remains elusive. The main goal of this work is to evaluate the applicability and functionality of the CRISPR-Cpf1 system in P. nicotinovorans. CRISPR-Cpf1 is a class 2 type V CRISPR system known to work in the closely related Corynebacterium glutamicum strains (Jiang et al., 2017). The draft genome of P. nicotinovorans was used to screen for the presence of incompatible CRISPR systems using CRISPRs web server (https://crispr.i2bc.paris-saclay.fr/). A number of 4 CRISPRs candidates were found on different contigs, but none were related to CRISPR-Cpf1. Hence, we concluded that the system might work in this strain and the pJYS3_crtYf plasmid containing a functional a CRISPR-Cpf1 system was electroporated into P. nicotinovorans. No transformants were obtained upon selection with kanamycin, indicating that the pJYS3 replicating origin might not be functional in P. nicotinovorans. Next, the CRISPR-Cpf1 genes from pJYS3_crtYf were amplified by PCR and ongoing work aims to clone these genes into a plasmid known to work in P. nicotinovorans – pART2 (Sandu et al., 2005).
Key words: CRISPR, genetic engineering, P. nicotinovorans
