Efficient genome editing ofMagnetospirillum magneticumAMB-1 by CRISPR-Cas9 system for analyzing magnetotactic behavior

Abstract

Magnetotactic bacteria are a diverse group of microorganisms with the ability to use geomagnetic fields for direction sensing. This magnetotactic behavior can help microorganisms move towards favorable habitats for optimal growth and reproduction. Highly efficient genomic editing is very useful for a comprehensive understanding of the magnetotactic mechanism at the molecular level. In this study, we adapted an engineered CRISPR-cas9 system for efficient inactivation of gene in a widely used magnetotactic bacteria model strain, Magnetospirillum magneticum AMB-1. By combining an engineered nuclease-deficient Cas9 and single-guide RNA, a CRISPR interference system was successfully developed to silence amb0994 expression. More importantly, we succeeded in the construction of a single amb0994 gene deletion mutant using CRISPR-Cas9 with approximate 60-fold high efficiency compared to classical homology double-crossing replacement procedure. This mutant synthesized normally the magnetosomes, but reacted quicker and with less time than the wild-type strain to abrupt magnetic field reversals. A dynamics simulation by modeling M. magneticum AMB-1 cell as an ellipsoid showed that the difference of the motions between wild and Δamb0994 is due to flagellar influence. The behavior observation being consistent with dynamics simulation indicated that Amb0994 is involved in the cellular response to magnetic torque change via controlling flagella. Besides the contribution to a better understanding of the magnetotaxis mechanism, this study demonstrates the CRISPR system as a useful genetic toolbox for high-efficiency genome editing in magnetotactic bacteria.

Magnetic genes: Studying the genetics of biomineralization in magnetotactic bacteria

“…However, genes identified in transposon-mutagenesis screens still require phenotypic validation with gene deletions. The development of more advanced geneediting technologies, like CRISPR, will be just as valuable for the study of MTB as it has been for other organisms [80,94]. For example, CRISPR interference (CRISPRi) can be used to knockdown multiple genes at a time more readily than traditional methods or to study essential genes by tuning their expression [95,96].…”