Individual gRNAs were cloned in an arrayed format using a Golden Gate-based approach. We designed primers encoding a gRNA with BbsI overhangs and an additional G for hU6 RNApolIII transcription (Forward: 5′-CACCG NNNNNNNNNNNNNNNNNNN-3′ and Reverse: 5′-AAACNNNNNNNNNNNNNNNNNNC -3′), annealed by boiling and slowly cooling to room temperature, and then ligated duplexes with a BbsI entry vector, BbsI-HF (NEB), T4 DNA ligase and buffer (NEB), 1X BSA (NEB) for 30X cutting (37°C) and ligating (16°C) cycles, before heat-shock transformation of DH5-α E. coli (NEB). All gRNA sequences used can be found online through the BE-view app.
T4 dna ligase and buffer
Manufactured by New England Biolabs
T4 DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in double-stranded DNA or RNA. The T4 DNA ligase buffer provides the optimal conditions for the enzymatic activity of T4 DNA ligase.
Automatically generated - may contain errors
Lab products found in correlation
Oligonucleotides for cloning, by Thermo Fisher Scientific (2 mentions)
Q5 hot start dna polymerase, by New England Biolabs (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Gotaq green master mix, by Promega (2 mentions)
E coli dh5α, by New England Biolabs (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Bbsi hf, by New England Biolabs (1 mentions)
T4 ligation buffer, by New England Biolabs (1 mentions)
Lenticrispr v2, by Addgene (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Top10 bacteria, by Thermo Fisher Scientific (1 mentions)
Plasmid safe, by Illumina (1 mentions)
Qiaquick pcr purification, by Qiagen (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Pcr supermix, by Thermo Fisher Scientific (1 mentions)
Flash gels, by Lonza (1 mentions)
Tae buffer, by New England Biolabs (1 mentions)
Hifi hotstart readymix, by Roche (1 mentions)
6 protocols using t4 dna ligase and buffer
1
Arrayed CRISPR gRNA Cloning Protocol
2
Inducible CRISPR-dCas9 Epigenome Editing
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Two single guide RNAs (sgRNAs) targeting each peak were designed using the online tool http://crispr.org (Supplementary Table 2 ). Two sgRNAs were designed for the CCND2 promoter as a positive control and the non-targeting sgRNA for Gal4 of the yeast S.cerevisiae as a negative control.
The vector used was the inducible Lenti-CRISPR-dCas9-KRABv2. The original LentiCRISPRv2 vector (Addgene 52961, USA) was modified and kindly supplied by Dr Niklas Feldhahn (Imperial College London). The plasmid was digested with Esp31 (New England Biolabs), removing a 2 kb stuffer.
The sgRNA oligos were phosphorylated and annealed using the T4 Ligation Buffer (New England Biolabs) and T4 PNK (New England Biolabs). The sgRNAs were then ligated with the digested plasmid using the T4 DNA ligase and buffer (New England Biolabs). Reactions were carried out following the Zhang Lab General Cloning Protocol (Addgene).
Sanger sequencing was used to confirm the exact sequence of the cloned sgRNA (GeneWiz Ltd UK).
The vector used was the inducible Lenti-CRISPR-dCas9-KRABv2. The original LentiCRISPRv2 vector (Addgene 52961, USA) was modified and kindly supplied by Dr Niklas Feldhahn (Imperial College London). The plasmid was digested with Esp31 (New England Biolabs), removing a 2 kb stuffer.
The sgRNA oligos were phosphorylated and annealed using the T4 Ligation Buffer (New England Biolabs) and T4 PNK (New England Biolabs). The sgRNAs were then ligated with the digested plasmid using the T4 DNA ligase and buffer (New England Biolabs). Reactions were carried out following the Zhang Lab General Cloning Protocol (Addgene).
Sanger sequencing was used to confirm the exact sequence of the cloned sgRNA (GeneWiz Ltd UK).
3
CRISPR Pooled Library Generation
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Single pGG vectors were generated using the same oligonucleotide ligation approach described above. Primer sequences can be found in S1 Table . Arrays were then generated using golden gate assembly. Briefly, 150 ng of each pGG-gRNA plasmid was combined with 150 ng of the appropriate pACPT vector, followed by addition of 1 uL of BsaI (New England BioLabs), 1 uL of T4 DNA Ligase and Buffer (New England BioLabs) and water to a total of 20 uL. Golden gate assembly was then carried out using the following thermocycling protocol: (37°C 5 minutes, 16°C 10 minutes) x10, 50°C 5 minutes, 80°C for 5 minutes and then cooled to 4°C. One uL of 25 mM ATP and 1 uL of Plasmid Safe (Epicentre) were then added to the reaction and incubated for 1 hour at 37°C. Assembled plasmids were then transformed into TOP10 bacteria (Thermo Fisher Scientific) and plated on spectinomycin selection plates with X-gal and mini-preps performed on white colonies using the GeneJET Plasmid Miniprep Kit (Life Technologies). The plasmids were then analyzed by Sanger sequencing to confirm proper gRNA array assembly.
4
Optimized Workflow for Bacterial Genome Annotation
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The bacterial strains and oligonucleotides used in this study are listed in Supplementary Tables S1 , S2 , respectively. oligonucleotides for cloning were ordered from Life Technologies 1 and oligonucleotides for next-generation sequencing libraries preparation were ordered from Integrated DNA Technologies.2 We used the GoTaq® Green Master Mix (Promega) for colony PCRs; and Q5 hot start DNA polymerase (New England Biolabs) for all other PCR reactions. DNA fragments were purified with the QIAquick PCR purification or Gel extraction kit (Qiagen). T4 DNA ligase and buffer were purchased from NEB. Plasmid and genomic DNA isolations were carried out with the QIAprep Spin Miniprep Kit and the DNeasy Blood & Tissue Kit (Qiagen), respectively.
The DvH genome annotation used in this study includes protein-coding genes that are not in the current version in GenBank (GCF_000195755.1). These additional genes were identified by transcriptomics and proteomics evidence (Price et al., 2011 (link)), and each starts with the systematic name “DORF.” These annotations are included in the DvH information at MicrobesOnline (Dehal et al., 2010 (link)).
The genes neighborhood comparison shown in this study was achieved using the BioCyc Database collection and the Ensembl Bacteria browser (Karp et al., 2019 (link); Howe et al., 2020 (link)).
The DvH genome annotation used in this study includes protein-coding genes that are not in the current version in GenBank (GCF_000195755.1). These additional genes were identified by transcriptomics and proteomics evidence (Price et al., 2011 (link)), and each starts with the systematic name “DORF.” These annotations are included in the DvH information at MicrobesOnline (Dehal et al., 2010 (link)).
The genes neighborhood comparison shown in this study was achieved using the BioCyc Database collection and the Ensembl Bacteria browser (Karp et al., 2019 (link); Howe et al., 2020 (link)).
5
Culturing and Molecular Techniques for P. gingivalis
6
Bacterial Strain and Molecular Techniques
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The bacterial strains and oligonucleotides used in this study are listed in Supplementary Tables 4 and5, respectively. oligonucleotides for cloning were ordered from Life Technologies (www.thermofisher.com) and oligonucleotides for next-generation sequencing libraries preparation were ordered from Integrated DNA Technologies (www.IDT.com). We used the GoTaq® Green Master Mix (Promega) for colony PCRs; and Q5 hot start DNA polymerase (New England Biolabs) for all other PCR reactions. DNA fragments were purified with the QIAquick PCR purification or Gel extraction kit (Qiagen). T4 DNA ligase and buffer were purchased from NEB. Plasmid and genomic DNA isolations were carried out with the QIAprep Spin Miniprep Kit and the DNeasy Blood & Tissue Kit (Qiagen), respectively.
The DvH genome annotation used in this study includes protein-coding genes that are not in the current version in GenBank (GCF_000195755.1). These additional genes were identified by transcriptomics and proteomics evidence (Price et al., 2011) , and each starts with the systematic name "DORF". These annotations are included in the DvH information at MicrobesOnline (Dehal et al., 2010) .
The genes neighborhood comparison shown in this study was achieved using the BioCyc Database collection and the Ensembl Bacteria browser (Howe et al., 2020; Karp et al., 2019) .
The DvH genome annotation used in this study includes protein-coding genes that are not in the current version in GenBank (GCF_000195755.1). These additional genes were identified by transcriptomics and proteomics evidence (Price et al., 2011) , and each starts with the systematic name "DORF". These annotations are included in the DvH information at MicrobesOnline (Dehal et al., 2010) .
The genes neighborhood comparison shown in this study was achieved using the BioCyc Database collection and the Ensembl Bacteria browser (Howe et al., 2020; Karp et al., 2019) .
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