Interference and editing of plasmids by a type I-F1 CRISPR-Cas system in different bacteria.

MARÍA CAROLINA MOLINA; CECILIA QUIROGA

Congreso; Congreso XVII SAMIGE 2022; 2022

SAMIGE

CRISPR-Cas systems of archaea and bacteria provide adaptive immunity against different mobile genetic elements, such as phages and plasmids. In the last years, there have been multiple efforts to transfer these systems to different organisms and redirect its activity towards specific genes leading to an augment in the gene editing field. The aim of this work was to investigate the potential use of a type I-F1 CRISPR-Cas system from Shewanella xiamenensis Sh95 as an endogenous DNA targeting tool against its own genome and to develop a heterologous system for other hosts. First, we designed synthetic CRISPR RNAs (crRNAs) guides cloned into pCDFDuet(StrR) carrying a spacer complementary to gfpmut3 (pG1) or a “mock” non-complementary spacer sequence (control, pG2). These crRNAs were introduced into S. xiamenensis Sh95 harboring the target vector pBKgfp(KmR). Very few co-transformants (1000 colonies/replicate were observed. Colonies with pG1 exhibited a reduction in the relative fluorescence measured by fluorimetry and confirmed under fluorescence microscopy. The loss of the target plasmid was confirmed by PCR. To test whether this system could be employed in an heterologous context, the structures csy1-csy2-csy3-csy4 (Csy complex coding genes) and cas2/3-csy1-csy2-csy3-csy4 (a variant which codes for the nuclease/helicase Cas2/3, were cloned into pRSFDuet(KmR) under control of the T7 inducible promoter. We studied its activity in the presence of pG1 and pG2 in E. coli BL21(DE3). When the target vector pCgfp(AmR) was transformed in the presence of gfpmut3 targeting guide (pG1) and the structure Csy complex+Cas2/3, 1000 colonies/replicate when Csy complex was employed. Fluorescence microscopy of some survival colonies carrying Csy+Cas2/3, pG1 and the target vector revealed the complete loss of GFP fluorescence. By PCR we could not amplify the surroundings of gfpmut3 likely because a large deletion event took place. However, with the control pG2 similar amounts of colonies with or without Cas2/3 were obtained and fluorescence intensity was not affected. This suggested that there was a leaky expression. We then assessed more tightly repressed conditions in E. coli BL21-AI and E. coli BL21(DE3)/pLysS. With E. coli BL21-AI, no colonies were obtained with Csy complex+Cas2/3, pG1 and the target vector present. Consistently, in E. coli BL21(DE3)/pLysS very few colonies were obtained in this same situation. Analysis of survival colonies by observation under fluorescence microscopy showed absence of fluorescence intensity. Sequence analysis of PCR products revealed a 243 pb deletion inside of gfpmut3 ORF. Our results demonstrate the feasibility and the practical use of the type I-F1 CRISPR-Cas system from S. xiamenensis Sh95 for gene editing in different bacteria as well as for DNA manipulation.