pcrispr sacb  (Addgene inc)

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

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: Overview of CRISPR/Cas9 genome editing strategy in Escherichia coli . The strain to be mutagenized [ E. coli BL21(DE3) ∆ompA ] is first transformed with the pCasRed plasmid expressing the λ Red (Exo, Beta, Gam) machinery, the Cas9 endonuclease, and tracrRNA. Subsequently, the strain is co-transformed with pCRISPR- SacB-gDNA , and a synthetic, mutation-inducing oligonucleotide [donor DNA (dDNA)]. The pCRISPR- SacB-gDNA plasmid encodes the gRNA that specifies the site of cleavage and the endonuclease Cas9 recognizes the gRNA together with the tracrRNA, which anneals to gRNA forming a three-component complex. After the base pairing of gRNA to the target site, the Cas9 mediates the chromosomal DNA double strand break ( upper panel ). The double strand break is repaired by λ Red-mediated homologous recombination taking place between the extremities of the cleaved chromosomal DNA and the donor DNA ( lower panel ). For the sequence of constitutive promoters P1 and P2 see ADDGENE #4287 ; for sequence of constitutive promoter P3 see ADDGENE #42875 and for P4 constitutive promoter sequence see ADDGENE #13036 . For the arabinose-inducible promoter pBAD see pKOBEG plasmid

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: CRISPR, Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Homologous Recombination, Sequencing

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: Selection of gDNAs for mutation of ompF , lpp and fecA genes. The grey bars are a schematic drawing of the genes lpp , fecA and ompF , and the black lines labelled with letters indicate the positions where the gRNAs transcribed from their corresponding gDNAs hybridize within each gene. gDNAs were cloned into pCRISPR, generating the plasmids reported in the Additional file : Table S1. The tables report the transformation efficiencies (CFU/μg) of each pCRISPR- gDNA in BL21(DE3) ∆ompA (pCasRed)

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: Selection, Mutagenesis, Clone Assay, Transformation Assay

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: Influence of type of donor DNA (dDNA) (Lg-ss-dDNAs, Ld-ss-dDNAs, ds-dDNA) length of dDNA, concentration of dDNA and size of deletion on mutagenesis efficiency at four chromosomal loci

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: Concentration Assay, Mutagenesis

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: Validation of 30 bp deletions on 78 genes. E. coli BL21(DE3)Δ ompA (pCasRed) or E. coli BL21(DE3)(pCasRed) were transformed with different mixtures of pCRISPR- SacB-gDNAs and 70-base dDNAs (either ssDNA or dsDNA) to mediate 30 bp deletion at one of the 78 selected gene loci ( y axis ). Ten colonies from each transformation were analyzed by PCR to identify those carrying the deletion. X axis indicates the percentage of mutants identified out of the total number of colonies analyzed. Bar height indicates the mutation frequency, while the presence of flat colored squares above gene names in each graph indicates that no mutants were identified out of ten colonies analyzed. Absence of bars or flat colored squares above gene names indicate that the gene mutation was not attempted for those specific genes in the experiment indicated in each bar graph. Red bars/squares indicate mutation experiments using Ld-ss-dDNAs; Blue bars/squares indicate mutation experiments using Lg-ss-dDNAs; Green bars/squares indicate mutation experiments using ds-dDNAs; bars with green downward diagonals indicate mutation experiments with ds-dDNAs in BL21(DE3)(pCasRed). a Bar graph representing mutation success rate using 46 Ld-ss-dDNAs ( red bars/squares ) and 32 Lg-ss-dDNAs ( blue bars/squares ). b Gene mutations that failed using the Ld-ss-DNAs (26 genes out of 46) and the Lg-ss-DNAs (4 genes out of 32) were re-attempted using ss-dDNAs targeting the opposite strands. The chart represents the mutation success rate of this second round of experiments. c The bar graph represents the mutation success rate in BL21(DE3) ΔompA (pCasRed) ( green bars ) and in BL21(DE3)(pCasRed) ( green downward diagonal bars ) using ds-dDNAs

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: Biomarker Discovery, Transformation Assay, Mutagenesis

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: Representation of the stepwise approach used to isolate strains carrying multiple mutations. Day 1: E. coli BL21(DE3) ∆ompA (pCasRed) was co-transformed with 1 μg/ml of pCRISPR- SacB-fecA_B and 10 μg/ml of donor fecA-120-∆30nt and transformant colonies were selected on LB agar plates supplemented with Cm (25 μg/ml) and Km (50 μg/ml). Day 2: Ten colonies were randomly selected and screened by PCR using primers designed to generate DNA fragments from mutated colonies of 200 bp. PCR products were analyzed on 2% agarose gels. One mutant clone was subsequently inoculated into 5 ml of LB supplemented with 5% sucrose and 25 μg/ml Cm. Day 3: The overnight culture was used to prepare competent cells, which were subsequently co-transformed with 1 μg/ml of pCRISPR- SacB-lpp_B and 10 μg of double strand donor DNA lpp-120-Δ237. Day 4: Ten colonies were randomly selected and screened by PCR using primers designed to generate DNA fragments from mutated colonies of 400 bp. PCR products were analyzed on 2% agarose gels

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: Transformation Assay, Mutagenesis

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: CRISPR/Cas-based protocol for simultaneous two-gene deletions E. coli BL21(DE3) ∆ompA (pCasRed) was co-transformed with either 100 ng pCRISPR- ompF_5′G - ompF_3′I plasmid and the two dDNAs ompF_5′G-120-∆30 and ompF_3′I-120-∆30 (10 μg each) ( a ) or with 100 ng pCRISPR- lpp_B - fecA_B and lpp_B-120-∆30 and fecA_B-120-∆30 dDNAs (10 μg each). b Transformant colonies were selected on LB agar plates supplemented with 25 μg/ml Cm and 50 μg/ml Km. Colony PCR was carried out using two different couple of primers to screen each genomic locus (indicated at the bottom of each gel) on a randomly selected number of colonies and the PCR products separated on 2% agarose gels. Asterisks indicate those colonies in which deletion occurred at both gene loci. The primer sequences used for PCR experiments are reported in Additional file : Table S5

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: CRISPR, Transformation Assay, Plasmid Preparation

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: Efficiency of simultaneous two-loci mutagenesis (ompF 5′/ompF 3′ regions and fecA/lpp genes) using pCRISPR plasmids carrying REPEAT-gDNA 1 -REPEAT-gDNA 2 -REPEAT cassette

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: Mutagenesis

Journal: Microbial Cell Factories

Article Title: Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

doi: 10.1186/s12934-017-0681-1

Figure Lengend Snippet: pCRISPR- SacB-gDNA plasmid curing using 5% sucrose containing medium. A single colony from E. coli BL21(DE3)Δ ompA strain carrying both the pCasRed (Cm resistance) and pCRISPR- SacB-gDNA (Km resistance) was grown at 37 °C in LB-medium containing 5% sucrose and 25 μg/ml Cm. After 14 h growth, 100 μl of culture were plated on LB-agar plates containing either Cm (25 μg/ml) + Km (50 μg/ml) or Cm (25 μg/ml) alone. The loss of pCRISPR- SacB-gDNA plasmid was verified by 1.5% agarose gel analysis of plasmids extracted from bacteria directly collected from the Cm-containing agar plate. As control, the same colony was grown in the absence of 5% sucrose and plasmid extraction was carried out from bacteria collected from LB-agar plate containing 25 μg/ml Cm

Article Snippet: The pCRISPR- SacB plasmid, derived from pCRISPR plasmid (ADDGENE #42875, [ ]), where a kanamycin resistance gene (Km R ) is fused to the sacB gene encoding the Bacillus subtilis levansucrase, carries the synthetic DNA fragment (gDNA) coding for the guide RNA necessary to drive the Cas9-dependent double stranded break at the desired site of the bacterial genome.

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Bacteria, Control, Extraction