Table 1.
Description of strains and plasmids.
| Strain | Organism | Description | Accession Number | Reference or source |
|---|---|---|---|---|
| DSM1313 | Clostridium thermocellum | wild type | CP002416 | DSMZ |
| LL1299 | Clostridium thermocellum | DSM1313 ΔhptΔ0478 | ||
| LL1584 | Clostridium thermocellum | LL1299 Peno-Cas operon | SRP164871 | This study |
| LL1585 | Clostridium thermocellum | LL1299 P#0815-Cas operon | SRP164872 | This study |
| LL1586 | Clostridium thermocellum | LL1299 PTsac#0068-Cas operon | SRP164875 | This study |
| LL1587 | Clostridium thermocellum | LL1299 PTsac#2130-Cas operon | SRP164873 | This study |
| LL1588 | Clostridium thermocellum | LL1299 PTsac#0530-Cas operon | SRP164874 | This study |
| 61 | Clostridium thermocellum | LL1586 one-step escape mutant, cas5 mutation | SRP164861 | This study |
| 62 | Clostridium thermocellum | LL1586 one-step escape mutant, cas3 mutation | SRP164864 | This study |
| 63 | Clostridium thermocellum | LL1586 one-step escape mutant, cas8 mutation | SRP164863 | This study |
| 67 | Clostridium thermocellum | LL1586 one-step escape mutant, cas5 mutation | SRP164866 | This study |
| 70 | Clostridium thermocellum | LL1586 one-step escape mutant, cas5 mutation | SRP164865 | This study |
| 71 | Clostridium thermocellum | LL1586 one-step escape mutant, cas8 mutation | SRP164867 | This study |
| 72 |
Clostridium thermocellum |
LL1586 one-step escape mutant, cas8 mutation |
SRP164868 |
This study |
|
Plasmids |
Description |
crRNA/spacer sequence |
Reference or Source |
|
| pJEW54 | PTsac0068GeoCas9; PClo1313_2638non-target_sgRNA | TTCCCTGGTACCTAGGAACCCG | This study | |
| pJEW55 | PTsac0068GeoCas9; PClo1313_2638sgRNA#1 | gggcatatttgttctggtcaag | This study | |
| pJEW56 | PTsac0068GeoCas9; PClo1313_2638sgRNA#2 | tcgtttctttttccgtctgcaa | This study | |
| pJEW57 | PTsac0068GeoCas9; PClo1313_2638sgRNA#3 | CGGGTTGACTGTCAGGGCATCC | This study | |
| pJEW63 | PTsac0068ThermoCas9; PClo1313_2638non-target_sgRNA | TGTCATAGCGCTAGATCCGG | This study | |
| pJEW64 | PTsac0068ThermoCas9; PClo1313_2638sgRNA#1 | gcatatttgttctggtcaag | This study | |
| pJEW70 | PTsac0068AceCas9; PClo1313_2638non-target_sgRNA | CTTCCGCTGAGACTCCCCTTACAC | This study | |
| pJEW71 | PTsac0068AceCas9; PClo1313_2638sgRNA#1 | ggattagaccctaaaattgaatat | This study | |
| pJEW72 | PTsac0068AceCas9; PClo1313_2638 sgRNA#2 |
caagatacgggttgactgtcaggg | This study | |
| pJEW68 | PTsac0068GeoCas9; PClo1313_2638 non-target_sgRNA_HA | TTCCCTGGTACCTAGGAACCCG | This study | |
| pJEW69 | PTsac0068GeoCas9; PClo1313_2638_sgRNA#1_HA | gggcatatttgttctggtcaag | This study | |
| pJEW84 | PTsac0068GeoCas9n; PClo1313_2638 non-target_sgRNA_HA | TTCCCTGGTACCTAGGAACCCG | This study | |
| pJEW85 | PTsac0068GeoCas9n; PClo1313_2638sgRNA#1_HA | gggcatatttgttctggtcaag | This study | |
| pJEW112 | HA | This study | ||
| pJEW106 | PTsac530A.caldusBeta_Exo; HA | This study | ||
| pJEW107 | PTsac530C.stercorariumRecT_RecE; HA | This study | ||
| pJEW108 | PTsac530GeobacillusRecT_RecE; HA | This study | ||
| pJEW136 | PTsac530A.caldusBeta_Exo; HA_500bp | This study | ||
| pJEW137 | PTsac530A.caldusBeta_Exo; HA_50bp | This study | ||
| pJEW117 | PTsac0068GeoCas9; PClo1313_2638non-target_sgRNA | TTCCCTGGTACCTAGGAACCCG | This study | |
| pJEW111 | PTsac0068GeoCas9; PClo1313_2638sgRNA#1 | gggcatatttgttctggtcaag | This study | |
| pTY11B | PClo1313_1194 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pTY21B | PClo1313_1194 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pTY32B | PClo1313_1194 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pTY62B | PClo1313_1194 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pTY11C | PClo1313_2638 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pTY21C | PClo1313_2638 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pTY32C | PClo1313_2638 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pTY62C | PClo1313_2638 | ATAATGACATTTATGGTACTGTTGTGGTAATAGACGA | This study | |
| pDGO185N | pyrF targeting, deletion | CAAGTTTCATAAAACACCCTCATGCCTTCAAGGCCGT | This study | |
| pDGO186N | pyrF targeting, deletion | TGAGATTGTTGCGGAGTATGTTGAATCATGGGGTGAA | This study | |
| pDGO187N | pyrF target, deletion, no sgRNA control | none | This study | |
| pDGO186N–S1_nheI | pyrF target, stop codon insertion, target sgRNA | TGAGATTGTTGCGGAGTATGTTGAATCATGGGGTGAA | This study | |
| pDGO186N–S1_CS | pyrF non-target control, stop codon insertion, non-target control sgRNA | CTTGAAGGCATGAGGGTGTTTTATGAAACTTGCAAAT | This study | |
| pDGO186NX-S1_nheI | pyrF target, stop codon insertion | none | This study | |
| pDGO186NXR-S1_nheI | pyrF target, stop codon insertion, recombinase | none | This study | |
| pDGO186N-CS3neo | target sgRNA | TGAGATTGTTGCGGAGTATGTTGAATCATGGGGTGAA | This study | |
| pDGO186N-ContS-neo | control sgRNA | CTTGAAGGCATGAGGGTGTTTTATGAAACTTGCAAAT | This study |