CRISPR-Cas9-mediated genome editing in vancomycin-resistant Enterococcus faecium

Abstract

The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report, we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faeciumβ-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates.

Conditionally essential genes for survival during starvation in Enterococcus faecium E745

“…The transposon library in E. faecium E745 was previously described [ 38 ]. The vectors pWS3 [ 40 ], pVDM1001 [ 41 ], pCRE-Lox [ 42 ], pEF25 [ 43 ] and pGPA1 [ 38 ] were obtained from our laboratory’s culture collection. Genomic DNA isolation was performed using the Wizard Genomic DNA Purification kit (Promega).…”

“…The usp deletion mutant was created using CRISPR-Cas9-mediated genome editing as previously described [ 41 ]. In short, a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) targeting usp was inserted into pVDM1001 by annealing oVDM2001 and oVDM2002 together and ligating this DNA fragment into BsaI-digested pVDM1001 creating pVDM-x usp .…”

Conditionally essential genes for survival during starvation inEnterococcus faeciumE745

“…The transposon library in E. faecium E745 was previously described [45]. The vectors pWS3 [47], pVDM1001 [48], pCRE-Lox [49], pEF25 [50] and pGPA1 [45] were obtained from our laboratory’s culture collection. Genomic DNA isolation was performed using the Wizard Genomic DNA Purification kit (Promega).…”

“…The usp deletion mutant was created using CRISPR-Cas9-mediated genome editing as previously described [48]. In short, a CRISPR targeting usp was inserted into pVDM1001 by annealing oVDM2001 and oVDM2002 together and ligating this DNA fragment into BsaI-digested pVDM1001 creating pVDM-x usp .…”

Prediction of off-target effects of the CRISPR/Cas9 system for design of sgRNA

“…The CRISPR/Cas9 system was initially discovered as the "immune system" for bacteria and archaea, which uses this strategy to fight against viruses that inject DNA into them [6][7][8][9] . The system has a prominent aspect as it only requires designing a singleguided RNA (sgRNA), which usually consists of twenty base pairs, that is capable of leading the Cas9 enzyme to bind to the site where the DNA sequence needs to be modified [10][11][12][13][14] . The sgRNA also helps identify with a specific sequence, called the protospacer adjacent motif (PAM), which is crucial in locating the segment of gene which needs modifications [15][16][17][18] .…”