pcrispr sacb gdna  (New England Biolabs)

Journal: Bio-protocol

Article Title: Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli

doi: 10.21769/BioProtoc.2688

Figure Lengend Snippet: A shows all three main components required for genome editing in E. coli: 1) the pCasRed plasmid expressing the λ Red (Exo, Beta, Gam) machinery, the Cas9 endonuclease, and the tracrRNA; 2) the pCRISPR-SacB-gDNA plasmid encoding the gRNA and the SacB gene; 3) a synthetic, double-stranded mutagenic oligonucleotide (ds-dDNA). After transformation, the tracrRNA anneals to the gRNA, which specifies the site of cleavage for Cas9 resulting in a three-component complex at the target locus, where the endonuclease activity mediates a chromosomal DNA double strand break (B). The double strand break is subsequently repaired by λ Red-mediated homologous recombination taking place between the extremities of the cleaved chromosomal DNA and the ds-dDNA (C).

Article Snippet: Pipette tips: GPR-10G (Mettler-Toledo, Rainin, catalog number: 17001862), GPR-250 (Mettler-Toledo, Rainin, catalog number: 17001861), GPR-1000 (Mettler-Toledo, Rainin, catalog number: 17001859) 50 ml tube (Corning, Falcon ® , catalog number: 352070) Primo ® Vacuum Filter Systems, 250 ml, 0.22 μm, PES (Euroclone, catalog number: EPVPE22250) Syringe filters PES 0.22 µm filters (Euroclone, catalog number: EPSPE2230) Petri dishes (Corning, Falcon ® , catalog number: 351007) MAX efficiency TM DH5α TM competent cells (Thermo Fisher Scientific, Invitrogen TM , catalog number: 18258012) Note: You can also use homemade competent cells of E. coli DH5α (see Step E1). pCasRed plasmid ( Zerbini et al. , 2017 ), modified pCas9 plasmid from Addgene (Addgene, catalog number: 42876) available upon request pCRISPR-SacB plasmid ( Zerbini et al. , 2017 ), modified pCRISPR plasmid from Addgene (Addgene, catalog number: 42875), available upon request T4 Polynucleotide Kinase (PNK) (New England Biolabs, catalog number: M0201S) T4 Polynucleotide Kinase (PNK) Reaction Buffer (New England Biolabs, catalog number: B0201S) Oligonucleotide 1 or gDNA forward (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B) Oligonucleotide 2 or gDNA reverse (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B) T4 ligase (New England Biolabs, catalog number: M0202S) 10x T4 ligase buffer (New England Biolabs, catalog number: B0202S) 1 M NaCl (Sigma-Aldrich, catalog number: S7653-1KG) Bsa I enzyme (New England Biolabs, catalog number: R0535L) Dpn I enzyme (New England Biolabs, catalog number: R0176S) Alkaline phosphatase, Calf Intestinal (CIP, New England Biolabs, catalog number: M0290S) Wizard ® SV Gel and PCR Clean-Up System (Promega, catalog number: A9282) QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106) ATLAS ClearSight DNA Stain (BioAtlas, catalog number: BH40501) Magnesium chloride (MgCl 2 ) (Sigma-Aldrich, catalog number: M8266-1KG) Note: See Recipes for preparation of the 100 mM working solution.

Techniques: Plasmid Preparation, Expressing, Transformation Assay, Activity Assay, Homologous Recombination

Journal: Bio-protocol

Article Title: Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli

doi: 10.21769/BioProtoc.2688

Figure Lengend Snippet: Step 1 includes the construction of the three main components needed for this protocol (see Figure 1). The E. coli strain you want to gene-edit (e.g., E. coli BL21(DE3)) has to be transformed with the pCasRed plasmid first (note asterisk, see Step E4a.i). The E. coli + pCasRed strain is the starting point for mutagenesis (Step 2), in which fresh electrocompetent cells are prepared (Step E3) and directly used for transformation (Step E4). The next day, a colony PCR is performed to identify positive knockout mutants (Steps E5 and E6), which are subsequently inoculated in liquid LB medium containing 5% sucrose for curing of the pCRISPR-SacB-gDNA used before (Step 3). Here, the protocol can be stopped or you continue with another round of mutagenesis following only the procedures from Step 2 highlighted in the dotted box. Legend: chloramphenicol (Cm), wild type (WT), positive mutant (+), escaper (-).

Article Snippet: Pipette tips: GPR-10G (Mettler-Toledo, Rainin, catalog number: 17001862), GPR-250 (Mettler-Toledo, Rainin, catalog number: 17001861), GPR-1000 (Mettler-Toledo, Rainin, catalog number: 17001859) 50 ml tube (Corning, Falcon ® , catalog number: 352070) Primo ® Vacuum Filter Systems, 250 ml, 0.22 μm, PES (Euroclone, catalog number: EPVPE22250) Syringe filters PES 0.22 µm filters (Euroclone, catalog number: EPSPE2230) Petri dishes (Corning, Falcon ® , catalog number: 351007) MAX efficiency TM DH5α TM competent cells (Thermo Fisher Scientific, Invitrogen TM , catalog number: 18258012) Note: You can also use homemade competent cells of E. coli DH5α (see Step E1). pCasRed plasmid ( Zerbini et al. , 2017 ), modified pCas9 plasmid from Addgene (Addgene, catalog number: 42876) available upon request pCRISPR-SacB plasmid ( Zerbini et al. , 2017 ), modified pCRISPR plasmid from Addgene (Addgene, catalog number: 42875), available upon request T4 Polynucleotide Kinase (PNK) (New England Biolabs, catalog number: M0201S) T4 Polynucleotide Kinase (PNK) Reaction Buffer (New England Biolabs, catalog number: B0201S) Oligonucleotide 1 or gDNA forward (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B) Oligonucleotide 2 or gDNA reverse (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B) T4 ligase (New England Biolabs, catalog number: M0202S) 10x T4 ligase buffer (New England Biolabs, catalog number: B0202S) 1 M NaCl (Sigma-Aldrich, catalog number: S7653-1KG) Bsa I enzyme (New England Biolabs, catalog number: R0535L) Dpn I enzyme (New England Biolabs, catalog number: R0176S) Alkaline phosphatase, Calf Intestinal (CIP, New England Biolabs, catalog number: M0290S) Wizard ® SV Gel and PCR Clean-Up System (Promega, catalog number: A9282) QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106) ATLAS ClearSight DNA Stain (BioAtlas, catalog number: BH40501) Magnesium chloride (MgCl 2 ) (Sigma-Aldrich, catalog number: M8266-1KG) Note: See Recipes for preparation of the 100 mM working solution.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Knock-Out

Journal: Bio-protocol

Article Title: Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli

doi: 10.21769/BioProtoc.2688

Figure Lengend Snippet: To design the guide DNA (gDNA) identify a PAM sequence (5’-NGG-3’, where N stands for any of the four nucleotides) near the 5’ end of the target gene. Then, select the 30 nucleotides upstream of the PAM region (protospacer). BLAST the entire sequence (protospacer + PAM), which is going to be the core of the forward oligonucleotide of your gDNA, against the genome of the target organism. To avoid Cas9 off-target effect, a ‘reliable’ gDNA should not have any homology among the 10-12 PAM-proximal nucleotides (seed region, in green) and less than 15% homology among the remaining protospacer when blasted against the rest of the genome. Finally, the chosen protospacer + PAM sequence is completed by the addition of five base pairs (in red) to facilitate the ligation for the subsequent cloning of the pCRISPR-SacB-gDNA, resulting in the forward oligonucleotide of the gDNA sequence. The reverse oligonucleotide of the gDNA is the reverse complement of the protospacer + PAM forward sequence with the addition of five base pairs (in red) as done for the forward oligonucleotide.

Article Snippet: Pipette tips: GPR-10G (Mettler-Toledo, Rainin, catalog number: 17001862), GPR-250 (Mettler-Toledo, Rainin, catalog number: 17001861), GPR-1000 (Mettler-Toledo, Rainin, catalog number: 17001859) 50 ml tube (Corning, Falcon ® , catalog number: 352070) Primo ® Vacuum Filter Systems, 250 ml, 0.22 μm, PES (Euroclone, catalog number: EPVPE22250) Syringe filters PES 0.22 µm filters (Euroclone, catalog number: EPSPE2230) Petri dishes (Corning, Falcon ® , catalog number: 351007) MAX efficiency TM DH5α TM competent cells (Thermo Fisher Scientific, Invitrogen TM , catalog number: 18258012) Note: You can also use homemade competent cells of E. coli DH5α (see Step E1). pCasRed plasmid ( Zerbini et al. , 2017 ), modified pCas9 plasmid from Addgene (Addgene, catalog number: 42876) available upon request pCRISPR-SacB plasmid ( Zerbini et al. , 2017 ), modified pCRISPR plasmid from Addgene (Addgene, catalog number: 42875), available upon request T4 Polynucleotide Kinase (PNK) (New England Biolabs, catalog number: M0201S) T4 Polynucleotide Kinase (PNK) Reaction Buffer (New England Biolabs, catalog number: B0201S) Oligonucleotide 1 or gDNA forward (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B) Oligonucleotide 2 or gDNA reverse (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B) T4 ligase (New England Biolabs, catalog number: M0202S) 10x T4 ligase buffer (New England Biolabs, catalog number: B0202S) 1 M NaCl (Sigma-Aldrich, catalog number: S7653-1KG) Bsa I enzyme (New England Biolabs, catalog number: R0535L) Dpn I enzyme (New England Biolabs, catalog number: R0176S) Alkaline phosphatase, Calf Intestinal (CIP, New England Biolabs, catalog number: M0290S) Wizard ® SV Gel and PCR Clean-Up System (Promega, catalog number: A9282) QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106) ATLAS ClearSight DNA Stain (BioAtlas, catalog number: BH40501) Magnesium chloride (MgCl 2 ) (Sigma-Aldrich, catalog number: M8266-1KG) Note: See Recipes for preparation of the 100 mM working solution.

Techniques: Sequencing, Ligation, Clone Assay