Table 1.
Strains and plasmids used in this study.
| Strain/plasmid | Genotype/Description | Source |
|---|---|---|
| Strains | ||
| E. coli DH5α | SupE44 ΔlacU169 (lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | Invitrogen |
| M. smegmatis mc2155 | High-frequency transformation mutant ATCC 607 | Snapper et al., 1990 |
| MsmdCas9 | M. smegmatis mc2155 with integrative plasmid containing dcas9Spy(pRH2502), kanR | This study |
| MsmdCas9_control | MsmdCas9 with non-integrative plasmid for inserting sgRNA (pRH2521). Empty vector control, kanR, hygR | This study |
| MsmdCas9_arr1 | MsmdCas9 with plasmid containing sgRNA targeting between−47 bp and −28 bp upstream arr start codon (pSG_arr1) kanR, hygR | This study |
| MsmdCas9_arr2 | MsmdCas9 with sgRNA targeting between−27 bp and −8 bp upstream arr start codon (pSG_arr2), kanR, hygR | This study |
| MsmdCas9_arr3 | MsmdCas9 with sgRNA targeting between +13 bp and +32 bp downstream of the arr start codon (pSG_arr3), kanR, hygR | This study |
| Plasmids | ||
| pRH2502 | Integrative plasmid derived from pTC-0X-1L, expressing dCas9Spy from an inducible tetRO promoter (uv15tetO) | Singh et al., 2016 |
| pRH2521 | Non-integrative plasmid derived from pTE-10M-0X, expressing sgRNA from an inducible tetRO promoter (Pmyc1tetO) | Singh et al., 2016 |
| pSG_arr1 | sgRNA targeting between −47 bp to −28 bp upstream of the arr start codon cloned into pRH2521 | This study |
| pSG_arr2 | sgRNA targeting between −27 bp to −8 bp upstream arr start codon cloned into pRH2521 | This study |
| pSG_arr3 | sgRNA targeting between +13 bp to +32 bp downstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo2 | sgRNA targeting between −50 bp to −38 bp upstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo3 | sgRNA targeting between −32 bp to −20 bp upstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo4 | sgRNA targeting between +66 bp to +78 bp downstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo5 | sgRNA targeting between −74 bp to −56 bp upstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo6 | sgRNA targeting between −56 bp to −38 bp upstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo7 | sgRNA targeting between +60 bp to +78 bp downstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo8 | sgRNA targeting between +166 bp to +184 bp downstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo9 | sgRNA targeting between −53 bp to −35 bp upstream of the arr start codon cloned into pRH2521 | This study |
| pSG_oligo10 | sgRNA targeting between −27 bp to −9 bp upstream of the arr start codon cloned into pRH2521 | This study |
The targeting region given does not include the 3 bp PAM site.