TABLE 1 .
Plasmids and strains
| Strain or plasmid name | Description | Reference and/or source |
|---|---|---|
| E. coli strain | ||
| EC1000 | Cloning host, providing repA in trans, for pLT06- and pGEM-T-Easy-derived plasmids | 63 |
| E. coli plasmids | ||
| pGEM T-Easy | Plasmid containing T-overhangs in MCS,a used for subcloning of DNA fragments for mutant generation in E. faecalis | Promega |
| pLT06 | Markerless exchange plasmid; confers chloramphenicol resistance | 58 |
| pWH03 | Derivative of pLT06 containing OG1RF_11778 and OG1RF_11789 for integration into neutral site on chromosome | 43 |
| pVP102 | Derivative of pLT06 to create markerless, in-frame deletion of CRISPR3-cas9 in T11RF | This study |
| pAS106 | Derivative of pLT06 to create deletion of spacer 6 in CRISPR3 locus of T11RF | This study |
| pVP105 | Derivative of pLT06 to change amino acid 7 of T11 CRISPR3 Cas9 from aspartic acid to alanine | This study |
| pG19 | Derivative of pWH03 to integrate the CRISPR1-cas9 gene, its native promoter, and predicted tracrRNA into the T11 chromosome between EFMG_00904 and EFMG_00905 | This study |
| pVP301 | Derivative of pWH03 to integrate the CRISPR3-cas9 gene, its native promoter, and predicted tracrRNA into the T11 chromosome between EFMG_00904 and EFMG_00905 | This study |
| pWH01 | Derivative of pLT06 to create markerless, in-frame deletion of OG1RF_11621-OG1RF_11622 in OG1SSp | 43 |
| pWH43 | Derivative of pWH03 to integrate OG1SSp OG1RF_11621-OG1RF_11622 and its native promoter into the chromosome between OG1RF_11778 and OG1RF_11789 | This study |
| pVP401 | Derivative of pGEM-T-Easy with 100-bp insert, including T11 CRISPR2 spacer 1 and the consensus CRISPR2 PAM | This study |
| pVP107 | Derivative of pLT06 to knock-in the T11 CRISPR2 spacer 1 sequence and consensus CRISPR2 PAM into the uvrB gene of pCF10 | This study |
| pVP402 | Derivative of pGEM-T-Easy with 100-bp insert, including T11 CRISPR2 spacer 1 and the consensus CRISPR3 PAM | This study |
| pVP108 | Derivative of pLT06 to knock-in T11 CRISPR2 spacer 1 and the consensus CRISPR3 PAM into the uvrB gene of pCF10 | This study |
| pVP109 | Derivative of pLT06 to change amino acid 601 of T11 CRISPR3 Cas9 from histidine to alanine | This study |
| E. faecalis strains | ||
| T11RF | Rifampin-fusidic acid-resistant derivative of T11 | 23 and this study |
| T11RFΔcas9 | T11RF CRISPR3-cas9 deletion mutant | This study |
| T11RFΔcas9+CR3 | T11RFΔcas9 mutant with chromosomal integration of CRISPR3 cas9 between EFMG_00904 and EFMG_00905 | This study |
| T11RFΔcas9+CR1 | T11RFΔcas9 mutant with chromosomal integration of CRISPR1 cas9 and the predicted CRISPR1 tracrRNA between EFMG_00904 and EFMG_00905 | This study |
| T11RFΔCR3S6 | T11RF with a deletion of CRISPR3 spacer 6 | This study |
| T11RFcas9D7A | T11RF with chromosomal mutation in the RuvC nuclease coding region of cas9 | This study |
| T11RFcas9H601A | T11RF with chromosomal mutation in the HNH nuclease coding region of cas9 | This study |
| T11RFcas9DM | T11RF with chromosomal mutations in the predicted RuvC and HNH nuclease coding regions of cas9 | This study |
| OG1SSp pAM714 | Spectinomycin-streptomycin-resistant derivative of OG1 harboring pAM714, an erythromycin (carried on Tn917)-resistant derivative of pAD1 | 44, 45 |
| VP701 | OG1SSp pAM714 EfaRFI deletion mutant | This study |
| WH702 | VP701 with chromosomal integration of EfaRFI (OG1RF_11621-OG1RF_11622) and its native promoter between OG1RF_11778 and OG1RF_11789 | This study |
| OG1SSp pCF10 | Spectinomycin-streptomycin-resistant derivative of OG1 harboring pCF10 encoding tetracycline resistance on Tn925 | 64 |
| VP703 | OG1SSp pCF10 EfaRFI deletion mutant | This study |
| OG1SSp pVP501 | OG1SSp pCF10 with insertion of T11 CRISPR2 spacer 1 and consensus CRISPR2 PAM into uvrB of pCF10 | This study |
| OG1SSp pVP502 | OG1SSp pCF10 with insertion of T11 CRISPR2 spacer1 and consensus CRISPR3 PAM into uvrB of pCF10 | This study |
a
MCS, multiple-cloning site.