Naslov
The effect of viability on CRISPR-Cas adaptation in Escherichia coli

Autori
Radovčić, Marin ; Ivančić-Baće, Ivana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Power of Microbes in Industry and Environment 2023 / Leboš Pavunc, Andreja - Zagreb, 2023, 10-10

Skup
Power of Microbes in Industry and Environment 2023

Mjesto i datum
Poreč, Hrvatska, 15.05.2023. - 18.05.2023

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
E. coli, CRISPR-Cas, Cas3, RecBCD

Sažetak
Insertion of new spacers into the CRISPR locus of many bacteria or archaea enables their protection from invading DNA elements. This process is called naïve adaptation and is catalysed by the Cas1-Cas2 complex only. Fragmentation of invading DNA prior to DNA insertion by Cas1-Cas2 is accomplished by RecBCD helicase activity supported by cellular single-stranded exonucleases. Consequently, naïve adaptation is strongly reduced in recB, recC or recD mutants. The assay for naïve adaptation requires ectopic expression of Cas1-Cas2 from the plasmid in cells lacking most of cas genes and monitoring of spacer acquisition by PCR in lysed bulk bacterial cultures after overnight growth. Spacers from certain regions of plasmids or chromosomes, such as the origin and terminus of replication or the CRISPR locus, were acquired more frequently. These regions are expected to contain more broken replication forks. The RecBCD enzyme is mainly involved in DNA repair of broken or damaged replication forks by homologous recombination, and in its absence, cell viability is greatly reduced. Therefore, in this work we wanted to test whether reduced viability affects the efficiency of adaptation in live recA recD and recB1080 cells compared to wt cells. To test this, for each strain we analysed the efficiency of adaptation, the origin of new spacers (chromosome or plasmid), the canonical or non-canonical PAM (protospacer adjacent motif), and the ability of the acquired spacers to induce autoimmunity in 10 cells that survived insertion of the new spacer. Our genetic results show that the efficiency of adaptation is not affected by the absence of recombination (reduced viability), but the number of acquired spacers with the canonical PAM or the origin of the new spacers is somewhat different than reported in the literature. Chromosomal spacers with the canonical PAM induced autoimmunity as expected.

Izvorni jezik
Engleski

POVEZANOST RADA