cas9 sgrna  (New England Biolabs)


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    EnGen sgRNA Synthesis Kit S pyogenes
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    EnGen sgRNA Synthesis Kit S pyogenes 20 rxns
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    E3322S
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    New England Biolabs cas9 sgrna
    EnGen sgRNA Synthesis Kit S pyogenes
    EnGen sgRNA Synthesis Kit S pyogenes 20 rxns
    https://www.bioz.com/result/cas9 sgrna/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas9 sgrna - by Bioz Stars, 2021-05
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    1) Product Images from "Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations"

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243781

    Gel electrophoresis results of mutation specific enrichment by TRACE on a phosphorothioated PCR product around the KRAS G12 locus. (A) Diagram of the PCR product designed around the KRAS G12 locus. (B) Results of TRACE performed on the 794 bp PCR product around KRAS G12 with the forward primer phosphorothioated. PCR product produced from normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease treatment. Lanes 3 and 4 show results of Cas9/sgRNA treated reactions with an sgRNA that is a perfect match to the normal variant PCR product without and with exonuclease treatment. Lanes 5 and 6 show results of Cas9/sgRNA treated reactions with an sgRNA that matches the KRAS G12D mutation, producing a mismatch in the first position prior to the PAM site without and with exonuclease treatment. (C) Diagram of the Cas9/sgRNA complex.
    Figure Legend Snippet: Gel electrophoresis results of mutation specific enrichment by TRACE on a phosphorothioated PCR product around the KRAS G12 locus. (A) Diagram of the PCR product designed around the KRAS G12 locus. (B) Results of TRACE performed on the 794 bp PCR product around KRAS G12 with the forward primer phosphorothioated. PCR product produced from normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease treatment. Lanes 3 and 4 show results of Cas9/sgRNA treated reactions with an sgRNA that is a perfect match to the normal variant PCR product without and with exonuclease treatment. Lanes 5 and 6 show results of Cas9/sgRNA treated reactions with an sgRNA that matches the KRAS G12D mutation, producing a mismatch in the first position prior to the PAM site without and with exonuclease treatment. (C) Diagram of the Cas9/sgRNA complex.

    Techniques Used: Nucleic Acid Electrophoresis, Mutagenesis, Polymerase Chain Reaction, Produced, Variant Assay

    Gel electrophoresis and qPCR results of enrichment from CAMP on five single targets and a 5-plex. (A) Gel electrophoresis results from CAMP targeting loci in KIT exon 18 (Lane 4), TP53 exon 10 (Lane 5), MET exon 19 (Lane 6), GNAQ exon 5 (Lane 7), and PDGFRA exon 18 (Lane 8). Results of the 5-plex of these targets is shown in Lane 9. Controls are shown in Lanes 1–3: Lane 1 shows reaction without Cas9/sgRNA but with UPS adapter and UPS primer, Lane 2 shows reaction without Cas9/sgRNA and without adapter but with UPS primer, and Lane 3 shows reaction without Cas9/sgRNA, UPS primer, and UPS adapter. (B) qPCR results from CAMP on the five individual targets and 5-plex. Enrichment was calculated by dividing the qPCR value for each target by an off-target qPCR value and then normalizing to a DNA standard sample. Enrichment for Samples 1–3 are averages for the five individual targets.
    Figure Legend Snippet: Gel electrophoresis and qPCR results of enrichment from CAMP on five single targets and a 5-plex. (A) Gel electrophoresis results from CAMP targeting loci in KIT exon 18 (Lane 4), TP53 exon 10 (Lane 5), MET exon 19 (Lane 6), GNAQ exon 5 (Lane 7), and PDGFRA exon 18 (Lane 8). Results of the 5-plex of these targets is shown in Lane 9. Controls are shown in Lanes 1–3: Lane 1 shows reaction without Cas9/sgRNA but with UPS adapter and UPS primer, Lane 2 shows reaction without Cas9/sgRNA and without adapter but with UPS primer, and Lane 3 shows reaction without Cas9/sgRNA, UPS primer, and UPS adapter. (B) qPCR results from CAMP on the five individual targets and 5-plex. Enrichment was calculated by dividing the qPCR value for each target by an off-target qPCR value and then normalizing to a DNA standard sample. Enrichment for Samples 1–3 are averages for the five individual targets.

    Techniques Used: Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction

    Gel electrophoresis showing single base discretion using cTRACE. (A) Demonstration of cTRACE on targets within KIT exon 18 and TP53 exon 10 in normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment with and without adapter, respectively, and amplified with KIT exon 18 chimeric primers. Lanes 3 and 7 show the results of cTRACE amplification with two perfectly matched primers for the two targets. Lanes 4 and 8 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch at the 3’-end. Lanes 5 and 9 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch in the second base from the 3’-end. Lanes 6 and 10 show the results of cTRACE amplification with one perfectly matched primer and one primer with two mismatches in the first and second position from the 3’-end. (B) Demonstration of cTRACE on KRAS exon 2 in normal human genomic DNA. Lane 1 shows enrichment found with a perfect matched primer and Lane 2 shows enrichment found with a single mismatch on the 3’-end of the primer that matches the KRAS G12D mutation.
    Figure Legend Snippet: Gel electrophoresis showing single base discretion using cTRACE. (A) Demonstration of cTRACE on targets within KIT exon 18 and TP53 exon 10 in normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment with and without adapter, respectively, and amplified with KIT exon 18 chimeric primers. Lanes 3 and 7 show the results of cTRACE amplification with two perfectly matched primers for the two targets. Lanes 4 and 8 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch at the 3’-end. Lanes 5 and 9 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch in the second base from the 3’-end. Lanes 6 and 10 show the results of cTRACE amplification with one perfectly matched primer and one primer with two mismatches in the first and second position from the 3’-end. (B) Demonstration of cTRACE on KRAS exon 2 in normal human genomic DNA. Lane 1 shows enrichment found with a perfect matched primer and Lane 2 shows enrichment found with a single mismatch on the 3’-end of the primer that matches the KRAS G12D mutation.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Mutagenesis

    Comparison of gel electrophoresis results for cCAMP and sequence specific PCR on five individual targets and a multiplex. (A) Gel electrophoresis results of cCAMP. Lanes 1 and 2 show controls without Cas9/sgRNA with and without UPS adapters; amplification is complete with UPS primers. Lanes 3–7 show the results of cCAMP targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 8 shows the five targets as a multiplex. Noted product lengths include 42 bp for the addition of the UPS adapters. (B) Gel results of sequence specific PCR designed to have the same melting temperature as the UPS component of the chimeric primers (63°C). (C) Gel results of sequence specific PCR designed to have the same melting temperature as the full chimeric primers (66°C). Sequence specific PCR were performed under same conditions as cCAMP (annealing temperature 65°C) and include a no template control in the first lane of each gel.
    Figure Legend Snippet: Comparison of gel electrophoresis results for cCAMP and sequence specific PCR on five individual targets and a multiplex. (A) Gel electrophoresis results of cCAMP. Lanes 1 and 2 show controls without Cas9/sgRNA with and without UPS adapters; amplification is complete with UPS primers. Lanes 3–7 show the results of cCAMP targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 8 shows the five targets as a multiplex. Noted product lengths include 42 bp for the addition of the UPS adapters. (B) Gel results of sequence specific PCR designed to have the same melting temperature as the UPS component of the chimeric primers (63°C). (C) Gel results of sequence specific PCR designed to have the same melting temperature as the full chimeric primers (66°C). Sequence specific PCR were performed under same conditions as cCAMP (annealing temperature 65°C) and include a no template control in the first lane of each gel.

    Techniques Used: Nucleic Acid Electrophoresis, Sequencing, Polymerase Chain Reaction, Multiplex Assay, Amplification

    cCAMP enrichment from a cfDNA model. (A) Gel electrophoresis of cCAMP with a cfDNA model input control reaction results. Lane 1 shows a control without Cas9/sgRNA, without UPS adapters, and is amplified with UPS primers. Lane 2 shows a control without Cas9/sgRNA with UPS adapter but no primers. Lane 3 shows a positive control without Cas9/sgRNA, with UPS adapters, and is amplified with a UPS primer. Lanes 4–8 show the results of the cCAMP protocol without the addition of Cas9/sgRNA complexes but with UPS adapters and chimeric primers for KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 9 shows the results of the multiplex without Cas9/sgRNA. (B) Gel electrophoresis of cCAMP with a cfDNA model input. Lanes 10–14 show the cCAMP procedure, including treatment with Cas9/sgRNA complexes targeting KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 15 shows results of the five targets as a multiplex. Note, the sizes listed only include the length required between the two Cas9/sgRNA complexes and do not include the length of the UPS adapter.
    Figure Legend Snippet: cCAMP enrichment from a cfDNA model. (A) Gel electrophoresis of cCAMP with a cfDNA model input control reaction results. Lane 1 shows a control without Cas9/sgRNA, without UPS adapters, and is amplified with UPS primers. Lane 2 shows a control without Cas9/sgRNA with UPS adapter but no primers. Lane 3 shows a positive control without Cas9/sgRNA, with UPS adapters, and is amplified with a UPS primer. Lanes 4–8 show the results of the cCAMP protocol without the addition of Cas9/sgRNA complexes but with UPS adapters and chimeric primers for KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 9 shows the results of the multiplex without Cas9/sgRNA. (B) Gel electrophoresis of cCAMP with a cfDNA model input. Lanes 10–14 show the cCAMP procedure, including treatment with Cas9/sgRNA complexes targeting KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 15 shows results of the five targets as a multiplex. Note, the sizes listed only include the length required between the two Cas9/sgRNA complexes and do not include the length of the UPS adapter.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Positive Control, Multiplex Assay

    Gel electrophoresis results of mutation specific enrichment by TRACE on a series of phosphorothioated PCR products. (A) TRACE on an 820 bp PCR product around the CFTR F2 sgRNA site with both primers phosphorothioated. (B) TRACE on the 820 bp PCR product with both primers phosphorylated. (C) TRACE on the 820 bp PCR product with the forward primer phosphorothioated and the reverse primer phosphorylated. (D) TRACE of the 820 bp PCR product with the forward primer phosphorylated and the reverse primer phosphorothioated. In each gel, (A)—(D), Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease. Lanes 3 and 4 show the results of Cas9/sgRNA treated reactions with a perfectly matched sgRNA without and with exonuclease treatment. Lanes 5 and 6 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the first position prior to the PAM site without and with exonuclease treatment. Lanes 7 and 8 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the third position prior to the PAM site without and with exonuclease treatment.
    Figure Legend Snippet: Gel electrophoresis results of mutation specific enrichment by TRACE on a series of phosphorothioated PCR products. (A) TRACE on an 820 bp PCR product around the CFTR F2 sgRNA site with both primers phosphorothioated. (B) TRACE on the 820 bp PCR product with both primers phosphorylated. (C) TRACE on the 820 bp PCR product with the forward primer phosphorothioated and the reverse primer phosphorylated. (D) TRACE of the 820 bp PCR product with the forward primer phosphorylated and the reverse primer phosphorothioated. In each gel, (A)—(D), Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease. Lanes 3 and 4 show the results of Cas9/sgRNA treated reactions with a perfectly matched sgRNA without and with exonuclease treatment. Lanes 5 and 6 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the first position prior to the PAM site without and with exonuclease treatment. Lanes 7 and 8 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the third position prior to the PAM site without and with exonuclease treatment.

    Techniques Used: Nucleic Acid Electrophoresis, Mutagenesis, Polymerase Chain Reaction

    Evidence for Cas9/sgRNA produced staggered cut. (A) Gel electrophoresis results of cCAMP with dA-tailing only before ligation. Lane 1 shows a control reaction without Cas9 with UPS adapter and the TP53 chimeric primers. Lanes 2–6 show results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (B) Gel electrophoresis results of cCAMP with dA-tailing module and added dCTP, dGTP and dTTP. Lanes 0 and 1 show control reactions without Cas9/sgRNA with and without UPS adapters with amplification complete with UPS primers. Lanes 2–6 show cCAMP results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (C) Analysis of components for the DNA pre-ligation preparation and ligation for TP53 exon 10. (D) Analysis of components for the DNA pre-ligation preparation and ligation for MET exon 19. For (D) Lane 0 shows a control without Cas9 with UPS adapter and MET exon 19 primers with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation. For (B) and (D) Lane 1 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation, Lane 2 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and KAPA HyperPlus Ligation, Lane 3 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and KAPA HyperPlus Ligation, Lane 4 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and NEBNext Ultra II Ligation, Lane 5 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and NEB ligation, and Lane 6 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and NEBNext Ultra II Ligation. In all samples, Klenow (exo-) was included in pre-ligation reaction.
    Figure Legend Snippet: Evidence for Cas9/sgRNA produced staggered cut. (A) Gel electrophoresis results of cCAMP with dA-tailing only before ligation. Lane 1 shows a control reaction without Cas9 with UPS adapter and the TP53 chimeric primers. Lanes 2–6 show results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (B) Gel electrophoresis results of cCAMP with dA-tailing module and added dCTP, dGTP and dTTP. Lanes 0 and 1 show control reactions without Cas9/sgRNA with and without UPS adapters with amplification complete with UPS primers. Lanes 2–6 show cCAMP results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (C) Analysis of components for the DNA pre-ligation preparation and ligation for TP53 exon 10. (D) Analysis of components for the DNA pre-ligation preparation and ligation for MET exon 19. For (D) Lane 0 shows a control without Cas9 with UPS adapter and MET exon 19 primers with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation. For (B) and (D) Lane 1 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation, Lane 2 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and KAPA HyperPlus Ligation, Lane 3 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and KAPA HyperPlus Ligation, Lane 4 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and NEBNext Ultra II Ligation, Lane 5 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and NEB ligation, and Lane 6 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and NEBNext Ultra II Ligation. In all samples, Klenow (exo-) was included in pre-ligation reaction.

    Techniques Used: Produced, Nucleic Acid Electrophoresis, Ligation, Multiplex Assay, Amplification

    2) Product Images from "Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells"

    Article Title: Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-019-0325-7

    CRISPR/Cas9 deletion of MTA1 in breast cancer cells. a . T7 endonuclease assay PCR results. Electropherogram of the T7 endonuclease digestion of MTA1 genomic PCR products visualized on an Agilent Bioanalyzer DNA 1000 Chip. Due to location of sgRNA target site, digestion of PCR products was predicted to generate the following fragments: wildtype 800 bp, sgRNA #1: 500 and 310 bp, sgRNA#3: 700 and 100 bp, and sgRNA #5: 760 and 40 bp. b . Western blot of MTA1 in MCF7 and MDA-MB-231 MTA1 knockout cells. GAPDH is included as an equal loading control. c . Cell proliferation assay of MCF7 and MDA-MB-231 knockout cells, compared to cells expressing an empty vector, n = 4 **** p
    Figure Legend Snippet: CRISPR/Cas9 deletion of MTA1 in breast cancer cells. a . T7 endonuclease assay PCR results. Electropherogram of the T7 endonuclease digestion of MTA1 genomic PCR products visualized on an Agilent Bioanalyzer DNA 1000 Chip. Due to location of sgRNA target site, digestion of PCR products was predicted to generate the following fragments: wildtype 800 bp, sgRNA #1: 500 and 310 bp, sgRNA#3: 700 and 100 bp, and sgRNA #5: 760 and 40 bp. b . Western blot of MTA1 in MCF7 and MDA-MB-231 MTA1 knockout cells. GAPDH is included as an equal loading control. c . Cell proliferation assay of MCF7 and MDA-MB-231 knockout cells, compared to cells expressing an empty vector, n = 4 **** p

    Techniques Used: CRISPR, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Western Blot, Multiple Displacement Amplification, Knock-Out, Proliferation Assay, Expressing, Plasmid Preparation

    3) Product Images from "Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations"

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243781

    Gel electrophoresis results of mutation specific enrichment by TRACE on a phosphorothioated PCR product around the KRAS G12 locus. (A) Diagram of the PCR product designed around the KRAS G12 locus. (B) Results of TRACE performed on the 794 bp PCR product around KRAS G12 with the forward primer phosphorothioated. PCR product produced from normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease treatment. Lanes 3 and 4 show results of Cas9/sgRNA treated reactions with an sgRNA that is a perfect match to the normal variant PCR product without and with exonuclease treatment. Lanes 5 and 6 show results of Cas9/sgRNA treated reactions with an sgRNA that matches the KRAS G12D mutation, producing a mismatch in the first position prior to the PAM site without and with exonuclease treatment. (C) Diagram of the Cas9/sgRNA complex.
    Figure Legend Snippet: Gel electrophoresis results of mutation specific enrichment by TRACE on a phosphorothioated PCR product around the KRAS G12 locus. (A) Diagram of the PCR product designed around the KRAS G12 locus. (B) Results of TRACE performed on the 794 bp PCR product around KRAS G12 with the forward primer phosphorothioated. PCR product produced from normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease treatment. Lanes 3 and 4 show results of Cas9/sgRNA treated reactions with an sgRNA that is a perfect match to the normal variant PCR product without and with exonuclease treatment. Lanes 5 and 6 show results of Cas9/sgRNA treated reactions with an sgRNA that matches the KRAS G12D mutation, producing a mismatch in the first position prior to the PAM site without and with exonuclease treatment. (C) Diagram of the Cas9/sgRNA complex.

    Techniques Used: Nucleic Acid Electrophoresis, Mutagenesis, Polymerase Chain Reaction, Produced, Variant Assay

    Gel electrophoresis and qPCR results of enrichment from CAMP on five single targets and a 5-plex. (A) Gel electrophoresis results from CAMP targeting loci in KIT exon 18 (Lane 4), TP53 exon 10 (Lane 5), MET exon 19 (Lane 6), GNAQ exon 5 (Lane 7), and PDGFRA exon 18 (Lane 8). Results of the 5-plex of these targets is shown in Lane 9. Controls are shown in Lanes 1–3: Lane 1 shows reaction without Cas9/sgRNA but with UPS adapter and UPS primer, Lane 2 shows reaction without Cas9/sgRNA and without adapter but with UPS primer, and Lane 3 shows reaction without Cas9/sgRNA, UPS primer, and UPS adapter. (B) qPCR results from CAMP on the five individual targets and 5-plex. Enrichment was calculated by dividing the qPCR value for each target by an off-target qPCR value and then normalizing to a DNA standard sample. Enrichment for Samples 1–3 are averages for the five individual targets.
    Figure Legend Snippet: Gel electrophoresis and qPCR results of enrichment from CAMP on five single targets and a 5-plex. (A) Gel electrophoresis results from CAMP targeting loci in KIT exon 18 (Lane 4), TP53 exon 10 (Lane 5), MET exon 19 (Lane 6), GNAQ exon 5 (Lane 7), and PDGFRA exon 18 (Lane 8). Results of the 5-plex of these targets is shown in Lane 9. Controls are shown in Lanes 1–3: Lane 1 shows reaction without Cas9/sgRNA but with UPS adapter and UPS primer, Lane 2 shows reaction without Cas9/sgRNA and without adapter but with UPS primer, and Lane 3 shows reaction without Cas9/sgRNA, UPS primer, and UPS adapter. (B) qPCR results from CAMP on the five individual targets and 5-plex. Enrichment was calculated by dividing the qPCR value for each target by an off-target qPCR value and then normalizing to a DNA standard sample. Enrichment for Samples 1–3 are averages for the five individual targets.

    Techniques Used: Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction

    Gel electrophoresis showing single base discretion using cTRACE. (A) Demonstration of cTRACE on targets within KIT exon 18 and TP53 exon 10 in normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment with and without adapter, respectively, and amplified with KIT exon 18 chimeric primers. Lanes 3 and 7 show the results of cTRACE amplification with two perfectly matched primers for the two targets. Lanes 4 and 8 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch at the 3’-end. Lanes 5 and 9 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch in the second base from the 3’-end. Lanes 6 and 10 show the results of cTRACE amplification with one perfectly matched primer and one primer with two mismatches in the first and second position from the 3’-end. (B) Demonstration of cTRACE on KRAS exon 2 in normal human genomic DNA. Lane 1 shows enrichment found with a perfect matched primer and Lane 2 shows enrichment found with a single mismatch on the 3’-end of the primer that matches the KRAS G12D mutation.
    Figure Legend Snippet: Gel electrophoresis showing single base discretion using cTRACE. (A) Demonstration of cTRACE on targets within KIT exon 18 and TP53 exon 10 in normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment with and without adapter, respectively, and amplified with KIT exon 18 chimeric primers. Lanes 3 and 7 show the results of cTRACE amplification with two perfectly matched primers for the two targets. Lanes 4 and 8 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch at the 3’-end. Lanes 5 and 9 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch in the second base from the 3’-end. Lanes 6 and 10 show the results of cTRACE amplification with one perfectly matched primer and one primer with two mismatches in the first and second position from the 3’-end. (B) Demonstration of cTRACE on KRAS exon 2 in normal human genomic DNA. Lane 1 shows enrichment found with a perfect matched primer and Lane 2 shows enrichment found with a single mismatch on the 3’-end of the primer that matches the KRAS G12D mutation.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Mutagenesis

    Comparison of gel electrophoresis results for cCAMP and sequence specific PCR on five individual targets and a multiplex. (A) Gel electrophoresis results of cCAMP. Lanes 1 and 2 show controls without Cas9/sgRNA with and without UPS adapters; amplification is complete with UPS primers. Lanes 3–7 show the results of cCAMP targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 8 shows the five targets as a multiplex. Noted product lengths include 42 bp for the addition of the UPS adapters. (B) Gel results of sequence specific PCR designed to have the same melting temperature as the UPS component of the chimeric primers (63°C). (C) Gel results of sequence specific PCR designed to have the same melting temperature as the full chimeric primers (66°C). Sequence specific PCR were performed under same conditions as cCAMP (annealing temperature 65°C) and include a no template control in the first lane of each gel.
    Figure Legend Snippet: Comparison of gel electrophoresis results for cCAMP and sequence specific PCR on five individual targets and a multiplex. (A) Gel electrophoresis results of cCAMP. Lanes 1 and 2 show controls without Cas9/sgRNA with and without UPS adapters; amplification is complete with UPS primers. Lanes 3–7 show the results of cCAMP targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 8 shows the five targets as a multiplex. Noted product lengths include 42 bp for the addition of the UPS adapters. (B) Gel results of sequence specific PCR designed to have the same melting temperature as the UPS component of the chimeric primers (63°C). (C) Gel results of sequence specific PCR designed to have the same melting temperature as the full chimeric primers (66°C). Sequence specific PCR were performed under same conditions as cCAMP (annealing temperature 65°C) and include a no template control in the first lane of each gel.

    Techniques Used: Nucleic Acid Electrophoresis, Sequencing, Polymerase Chain Reaction, Multiplex Assay, Amplification

    cCAMP enrichment from a cfDNA model. (A) Gel electrophoresis of cCAMP with a cfDNA model input control reaction results. Lane 1 shows a control without Cas9/sgRNA, without UPS adapters, and is amplified with UPS primers. Lane 2 shows a control without Cas9/sgRNA with UPS adapter but no primers. Lane 3 shows a positive control without Cas9/sgRNA, with UPS adapters, and is amplified with a UPS primer. Lanes 4–8 show the results of the cCAMP protocol without the addition of Cas9/sgRNA complexes but with UPS adapters and chimeric primers for KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 9 shows the results of the multiplex without Cas9/sgRNA. (B) Gel electrophoresis of cCAMP with a cfDNA model input. Lanes 10–14 show the cCAMP procedure, including treatment with Cas9/sgRNA complexes targeting KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 15 shows results of the five targets as a multiplex. Note, the sizes listed only include the length required between the two Cas9/sgRNA complexes and do not include the length of the UPS adapter.
    Figure Legend Snippet: cCAMP enrichment from a cfDNA model. (A) Gel electrophoresis of cCAMP with a cfDNA model input control reaction results. Lane 1 shows a control without Cas9/sgRNA, without UPS adapters, and is amplified with UPS primers. Lane 2 shows a control without Cas9/sgRNA with UPS adapter but no primers. Lane 3 shows a positive control without Cas9/sgRNA, with UPS adapters, and is amplified with a UPS primer. Lanes 4–8 show the results of the cCAMP protocol without the addition of Cas9/sgRNA complexes but with UPS adapters and chimeric primers for KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 9 shows the results of the multiplex without Cas9/sgRNA. (B) Gel electrophoresis of cCAMP with a cfDNA model input. Lanes 10–14 show the cCAMP procedure, including treatment with Cas9/sgRNA complexes targeting KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 15 shows results of the five targets as a multiplex. Note, the sizes listed only include the length required between the two Cas9/sgRNA complexes and do not include the length of the UPS adapter.

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Positive Control, Multiplex Assay

    Gel electrophoresis results of mutation specific enrichment by TRACE on a series of phosphorothioated PCR products. (A) TRACE on an 820 bp PCR product around the CFTR F2 sgRNA site with both primers phosphorothioated. (B) TRACE on the 820 bp PCR product with both primers phosphorylated. (C) TRACE on the 820 bp PCR product with the forward primer phosphorothioated and the reverse primer phosphorylated. (D) TRACE of the 820 bp PCR product with the forward primer phosphorylated and the reverse primer phosphorothioated. In each gel, (A)—(D), Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease. Lanes 3 and 4 show the results of Cas9/sgRNA treated reactions with a perfectly matched sgRNA without and with exonuclease treatment. Lanes 5 and 6 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the first position prior to the PAM site without and with exonuclease treatment. Lanes 7 and 8 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the third position prior to the PAM site without and with exonuclease treatment.
    Figure Legend Snippet: Gel electrophoresis results of mutation specific enrichment by TRACE on a series of phosphorothioated PCR products. (A) TRACE on an 820 bp PCR product around the CFTR F2 sgRNA site with both primers phosphorothioated. (B) TRACE on the 820 bp PCR product with both primers phosphorylated. (C) TRACE on the 820 bp PCR product with the forward primer phosphorothioated and the reverse primer phosphorylated. (D) TRACE of the 820 bp PCR product with the forward primer phosphorylated and the reverse primer phosphorothioated. In each gel, (A)—(D), Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease. Lanes 3 and 4 show the results of Cas9/sgRNA treated reactions with a perfectly matched sgRNA without and with exonuclease treatment. Lanes 5 and 6 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the first position prior to the PAM site without and with exonuclease treatment. Lanes 7 and 8 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the third position prior to the PAM site without and with exonuclease treatment.

    Techniques Used: Nucleic Acid Electrophoresis, Mutagenesis, Polymerase Chain Reaction

    Evidence for Cas9/sgRNA produced staggered cut. (A) Gel electrophoresis results of cCAMP with dA-tailing only before ligation. Lane 1 shows a control reaction without Cas9 with UPS adapter and the TP53 chimeric primers. Lanes 2–6 show results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (B) Gel electrophoresis results of cCAMP with dA-tailing module and added dCTP, dGTP and dTTP. Lanes 0 and 1 show control reactions without Cas9/sgRNA with and without UPS adapters with amplification complete with UPS primers. Lanes 2–6 show cCAMP results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (C) Analysis of components for the DNA pre-ligation preparation and ligation for TP53 exon 10. (D) Analysis of components for the DNA pre-ligation preparation and ligation for MET exon 19. For (D) Lane 0 shows a control without Cas9 with UPS adapter and MET exon 19 primers with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation. For (B) and (D) Lane 1 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation, Lane 2 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and KAPA HyperPlus Ligation, Lane 3 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and KAPA HyperPlus Ligation, Lane 4 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and NEBNext Ultra II Ligation, Lane 5 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and NEB ligation, and Lane 6 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and NEBNext Ultra II Ligation. In all samples, Klenow (exo-) was included in pre-ligation reaction.
    Figure Legend Snippet: Evidence for Cas9/sgRNA produced staggered cut. (A) Gel electrophoresis results of cCAMP with dA-tailing only before ligation. Lane 1 shows a control reaction without Cas9 with UPS adapter and the TP53 chimeric primers. Lanes 2–6 show results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (B) Gel electrophoresis results of cCAMP with dA-tailing module and added dCTP, dGTP and dTTP. Lanes 0 and 1 show control reactions without Cas9/sgRNA with and without UPS adapters with amplification complete with UPS primers. Lanes 2–6 show cCAMP results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (C) Analysis of components for the DNA pre-ligation preparation and ligation for TP53 exon 10. (D) Analysis of components for the DNA pre-ligation preparation and ligation for MET exon 19. For (D) Lane 0 shows a control without Cas9 with UPS adapter and MET exon 19 primers with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation. For (B) and (D) Lane 1 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation, Lane 2 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and KAPA HyperPlus Ligation, Lane 3 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and KAPA HyperPlus Ligation, Lane 4 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and NEBNext Ultra II Ligation, Lane 5 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and NEB ligation, and Lane 6 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and NEBNext Ultra II Ligation. In all samples, Klenow (exo-) was included in pre-ligation reaction.

    Techniques Used: Produced, Nucleic Acid Electrophoresis, Ligation, Multiplex Assay, Amplification

    4) Product Images from "Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity"

    Article Title: Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity

    Journal: bioRxiv

    doi: 10.1101/685032

    Domain insertion into AcrIIC1 yields a highly potent Nme Cas9 inhibitor. ( a , b ) HEK 293T cells were co-transfected with vectors expressing Nme Cas9, the indicated Acr and sgRNAs targeting different genomic loci followed by T7 endonuclease assay. In a , Acr: Nme Cas9 vector ratio used during transfection was 1:1, while in b , the indicated, low Acr: Nme Cas9 vector ratios were used. Representative T7 gel images and corresponding quantification of indel frequencies are shown. Lines in plots show means, dots are individual data points for n = 4 (DHFR, AAVS1 and IL2RG locus) or n = 3 (F8 locus) independent experiments. Chim., AcrIIC1-mCherry chimeras in Supplementary Fig. 2. In., input band. T7, T7 cleavage fragments. ( c ) AcrIIC1-mCherry chimeras outperform wild-type AcrIIC1 and AcrIIC3. Cells were co-transfected with vectors encoding Nme Cas9, a firefly luciferase reporter and corresponding reporter-targeting sgRNA as well as the indicated Acr followed by luciferase assay. Bars indicate means, error bars the SD for n = 3 independent experiments. ( a-c ) N, negative (Cas 9 only ( a , b ) or reporter only ( c ) control (Ctrl.)). P, positive control (Cas9 + sgRNA ( a , b ) or reporter + Cas9 ( c )). * P
    Figure Legend Snippet: Domain insertion into AcrIIC1 yields a highly potent Nme Cas9 inhibitor. ( a , b ) HEK 293T cells were co-transfected with vectors expressing Nme Cas9, the indicated Acr and sgRNAs targeting different genomic loci followed by T7 endonuclease assay. In a , Acr: Nme Cas9 vector ratio used during transfection was 1:1, while in b , the indicated, low Acr: Nme Cas9 vector ratios were used. Representative T7 gel images and corresponding quantification of indel frequencies are shown. Lines in plots show means, dots are individual data points for n = 4 (DHFR, AAVS1 and IL2RG locus) or n = 3 (F8 locus) independent experiments. Chim., AcrIIC1-mCherry chimeras in Supplementary Fig. 2. In., input band. T7, T7 cleavage fragments. ( c ) AcrIIC1-mCherry chimeras outperform wild-type AcrIIC1 and AcrIIC3. Cells were co-transfected with vectors encoding Nme Cas9, a firefly luciferase reporter and corresponding reporter-targeting sgRNA as well as the indicated Acr followed by luciferase assay. Bars indicate means, error bars the SD for n = 3 independent experiments. ( a-c ) N, negative (Cas 9 only ( a , b ) or reporter only ( c ) control (Ctrl.)). P, positive control (Cas9 + sgRNA ( a , b ) or reporter + Cas9 ( c )). * P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase, Positive Control

    Related Articles

    other:

    Article Title: Lipid peroxidation regulates long-range wound detection through 5-lipoxygenase in zebrafish.
    Article Snippet: To generate templates for sgRNA transcription, gene specific oligonucleotides against zebrafish alox12 (ENSDARG00000069463) were designed using CHOPCHOP ( https://chopchop.cbu.uib.no ).

    Article Title: Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells
    Article Snippet: Successful editing was determined by the presence of T7EI cleaved products in the Cas9/sgRNA transduced cells compared to wildtype cells.

    Transfection:

    Article Title: Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity
    Article Snippet: Cells were co-transfected with 100, 133 or 160 ng of Acr vector and 100, 67 or 40 ng or all-in-one Cas9/sgRNA vector corresponding to Acr:Cas9 vector ratios of 1:1, 2:1 and 4:1, respectively, as indicated in the figures. .. Transfections for the initial screen of the chimeric AcrIIC1 variants (Supplementary Fig. 2) were performed with only 100 ng of total DNA per well, using a 1:1 ratio of Cas9/sgRNA and Acr vectors. ..

    Polymerase Chain Reaction:

    Article Title: CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis
    Article Snippet: Standard desalted sgRNA forward 60-nt and the 80-nt universal Cas9 reverse oligonucleotide primers (Supplemental Fig. ) were custom-synthesized (Sigma). .. To generate DNA templates for sgRNA synthesis, we used a PCR-based strategy to make linear double-stranded DNA templates for in vitro transcription . .. The two long, partially overlapping, forward and reverse oligonucleotides were annealed and used as PCR primers with a high fidelity Q5 DNA polymerase (New England BioLabs [NEB]; Supplemental Fig. ).

    Article Title: CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis
    Article Snippet: .. The nucleotide (nt) sequences used to generate PCR templates for sgRNA synthesis were designed with the protospacer adjacent motif (PAM) site removed and the two nucleotides at the 5′ end of the 20-nt target sequence were altered to GG when necessary in the oligonucleotide forward primer (Supplemental Fig. ) . .. Standard desalted sgRNA forward 60-nt and the 80-nt universal Cas9 reverse oligonucleotide primers (Supplemental Fig. ) were custom-synthesized (Sigma).

    In Vitro:

    Article Title: CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis
    Article Snippet: Standard desalted sgRNA forward 60-nt and the 80-nt universal Cas9 reverse oligonucleotide primers (Supplemental Fig. ) were custom-synthesized (Sigma). .. To generate DNA templates for sgRNA synthesis, we used a PCR-based strategy to make linear double-stranded DNA templates for in vitro transcription . .. The two long, partially overlapping, forward and reverse oligonucleotides were annealed and used as PCR primers with a high fidelity Q5 DNA polymerase (New England BioLabs [NEB]; Supplemental Fig. ).

    Produced:

    Article Title: CONTEXT-INDEPENDENT FUNCTION OF A CHROMATIN BOUNDARY IN VIVO
    Article Snippet: .. Multiple pairs of sgRNAs were designed on the target region ( , bottom) with Benchling ( https://www.benchling.com/ ) and were converted into EnGen-compatible DNA oligos (listed in ) using NEBioCalculator ( http://nebiocalculator.neb.com/#!/sgrna ). sgRNAs were produced using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB, E3322) following the manufacturer’s instructions. .. Two distinct pools of sgRNAs and Alt-R S. pyogenes HiFi Cas9 nuclease V3 (IDT) were assembled into Cas9 ribonucleoprotein complexes.

    Activity Assay:

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations
    Article Snippet: .. Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ]. .. We hypothesized that these staggered ends generated by Cas9/sgRNA are present during the cCAMP protocol and cause a loss of enrichment in several of the targets when the components for a fill-in repair are not provided before ligation.

    Sequencing:

    Article Title: CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis
    Article Snippet: .. The nucleotide (nt) sequences used to generate PCR templates for sgRNA synthesis were designed with the protospacer adjacent motif (PAM) site removed and the two nucleotides at the 5′ end of the 20-nt target sequence were altered to GG when necessary in the oligonucleotide forward primer (Supplemental Fig. ) . .. Standard desalted sgRNA forward 60-nt and the 80-nt universal Cas9 reverse oligonucleotide primers (Supplemental Fig. ) were custom-synthesized (Sigma).

    Generated:

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations
    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ]. .. We hypothesized that these staggered ends generated by Cas9/sgRNA are present during the cCAMP protocol and cause a loss of enrichment in several of the targets when the components for a fill-in repair are not provided before ligation. .. To investigate this hypothesis, the components for the pre-ligation and ligation steps were compared for TP53 exon 10, which showed amplification with both protocols , and for MET exon 19 which only showed amplification with the reagents purchased from Kapa Biosystems which had all four dNTPs ( ) present for repair.

    Ligation:

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations
    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ]. .. We hypothesized that these staggered ends generated by Cas9/sgRNA are present during the cCAMP protocol and cause a loss of enrichment in several of the targets when the components for a fill-in repair are not provided before ligation. .. To investigate this hypothesis, the components for the pre-ligation and ligation steps were compared for TP53 exon 10, which showed amplification with both protocols , and for MET exon 19 which only showed amplification with the reagents purchased from Kapa Biosystems which had all four dNTPs ( ) present for repair.

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    New England Biolabs cas9 sgrna
    <t>CRISPR/Cas9</t> deletion of MTA1 in breast cancer cells. a . T7 endonuclease assay PCR results. Electropherogram of the T7 endonuclease digestion of MTA1 genomic PCR products visualized on an Agilent Bioanalyzer DNA 1000 Chip. Due to location of <t>sgRNA</t> target site, digestion of PCR products was predicted to generate the following fragments: wildtype 800 bp, sgRNA #1: 500 and 310 bp, sgRNA#3: 700 and 100 bp, and sgRNA #5: 760 and 40 bp. b . Western blot of MTA1 in MCF7 and MDA-MB-231 MTA1 knockout cells. GAPDH is included as an equal loading control. c . Cell proliferation assay of MCF7 and MDA-MB-231 knockout cells, compared to cells expressing an empty vector, n = 4 **** p
    Cas9 Sgrna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs sgrna guided cas9
    PGRMC1 knockout using a <t>CRISPR/Cas9</t> approach. A. Schematic of the PGRMC1 <t>sgRNA/Cas9-expressing</t> lentiviral constructs for knocking out PGRMC1. Gray areas show the three candidate sgRNA sequences in the exon-1 of the PGRMC1 gene, of which two were used for the CRISPR/Cas9 constructs. B. A representative DNA gel of control and PGRMC1 knockout (clones 38 and 207) NSC34 cells verifying the Cas9 cleavage of the genomic DNA. Control refers to the NSC34 cells transfected with the control vector expressing Cas9 but not an sgRNA. C. Western blotting detection of PGRMC1 in control and PGRMC1 knockout (clones 38 and 207) NSC34 cells.
    Sgrna Guided Cas9, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CRISPR/Cas9 deletion of MTA1 in breast cancer cells. a . T7 endonuclease assay PCR results. Electropherogram of the T7 endonuclease digestion of MTA1 genomic PCR products visualized on an Agilent Bioanalyzer DNA 1000 Chip. Due to location of sgRNA target site, digestion of PCR products was predicted to generate the following fragments: wildtype 800 bp, sgRNA #1: 500 and 310 bp, sgRNA#3: 700 and 100 bp, and sgRNA #5: 760 and 40 bp. b . Western blot of MTA1 in MCF7 and MDA-MB-231 MTA1 knockout cells. GAPDH is included as an equal loading control. c . Cell proliferation assay of MCF7 and MDA-MB-231 knockout cells, compared to cells expressing an empty vector, n = 4 **** p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells

    doi: 10.1186/s12964-019-0325-7

    Figure Lengend Snippet: CRISPR/Cas9 deletion of MTA1 in breast cancer cells. a . T7 endonuclease assay PCR results. Electropherogram of the T7 endonuclease digestion of MTA1 genomic PCR products visualized on an Agilent Bioanalyzer DNA 1000 Chip. Due to location of sgRNA target site, digestion of PCR products was predicted to generate the following fragments: wildtype 800 bp, sgRNA #1: 500 and 310 bp, sgRNA#3: 700 and 100 bp, and sgRNA #5: 760 and 40 bp. b . Western blot of MTA1 in MCF7 and MDA-MB-231 MTA1 knockout cells. GAPDH is included as an equal loading control. c . Cell proliferation assay of MCF7 and MDA-MB-231 knockout cells, compared to cells expressing an empty vector, n = 4 **** p

    Article Snippet: Successful editing was determined by the presence of T7EI cleaved products in the Cas9/sgRNA transduced cells compared to wildtype cells.

    Techniques: CRISPR, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Western Blot, Multiple Displacement Amplification, Knock-Out, Proliferation Assay, Expressing, Plasmid Preparation

    Gel electrophoresis results of mutation specific enrichment by TRACE on a phosphorothioated PCR product around the KRAS G12 locus. (A) Diagram of the PCR product designed around the KRAS G12 locus. (B) Results of TRACE performed on the 794 bp PCR product around KRAS G12 with the forward primer phosphorothioated. PCR product produced from normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease treatment. Lanes 3 and 4 show results of Cas9/sgRNA treated reactions with an sgRNA that is a perfect match to the normal variant PCR product without and with exonuclease treatment. Lanes 5 and 6 show results of Cas9/sgRNA treated reactions with an sgRNA that matches the KRAS G12D mutation, producing a mismatch in the first position prior to the PAM site without and with exonuclease treatment. (C) Diagram of the Cas9/sgRNA complex.

    Journal: PLoS ONE

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    doi: 10.1371/journal.pone.0243781

    Figure Lengend Snippet: Gel electrophoresis results of mutation specific enrichment by TRACE on a phosphorothioated PCR product around the KRAS G12 locus. (A) Diagram of the PCR product designed around the KRAS G12 locus. (B) Results of TRACE performed on the 794 bp PCR product around KRAS G12 with the forward primer phosphorothioated. PCR product produced from normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease treatment. Lanes 3 and 4 show results of Cas9/sgRNA treated reactions with an sgRNA that is a perfect match to the normal variant PCR product without and with exonuclease treatment. Lanes 5 and 6 show results of Cas9/sgRNA treated reactions with an sgRNA that matches the KRAS G12D mutation, producing a mismatch in the first position prior to the PAM site without and with exonuclease treatment. (C) Diagram of the Cas9/sgRNA complex.

    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ].

    Techniques: Nucleic Acid Electrophoresis, Mutagenesis, Polymerase Chain Reaction, Produced, Variant Assay

    Gel electrophoresis and qPCR results of enrichment from CAMP on five single targets and a 5-plex. (A) Gel electrophoresis results from CAMP targeting loci in KIT exon 18 (Lane 4), TP53 exon 10 (Lane 5), MET exon 19 (Lane 6), GNAQ exon 5 (Lane 7), and PDGFRA exon 18 (Lane 8). Results of the 5-plex of these targets is shown in Lane 9. Controls are shown in Lanes 1–3: Lane 1 shows reaction without Cas9/sgRNA but with UPS adapter and UPS primer, Lane 2 shows reaction without Cas9/sgRNA and without adapter but with UPS primer, and Lane 3 shows reaction without Cas9/sgRNA, UPS primer, and UPS adapter. (B) qPCR results from CAMP on the five individual targets and 5-plex. Enrichment was calculated by dividing the qPCR value for each target by an off-target qPCR value and then normalizing to a DNA standard sample. Enrichment for Samples 1–3 are averages for the five individual targets.

    Journal: PLoS ONE

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    doi: 10.1371/journal.pone.0243781

    Figure Lengend Snippet: Gel electrophoresis and qPCR results of enrichment from CAMP on five single targets and a 5-plex. (A) Gel electrophoresis results from CAMP targeting loci in KIT exon 18 (Lane 4), TP53 exon 10 (Lane 5), MET exon 19 (Lane 6), GNAQ exon 5 (Lane 7), and PDGFRA exon 18 (Lane 8). Results of the 5-plex of these targets is shown in Lane 9. Controls are shown in Lanes 1–3: Lane 1 shows reaction without Cas9/sgRNA but with UPS adapter and UPS primer, Lane 2 shows reaction without Cas9/sgRNA and without adapter but with UPS primer, and Lane 3 shows reaction without Cas9/sgRNA, UPS primer, and UPS adapter. (B) qPCR results from CAMP on the five individual targets and 5-plex. Enrichment was calculated by dividing the qPCR value for each target by an off-target qPCR value and then normalizing to a DNA standard sample. Enrichment for Samples 1–3 are averages for the five individual targets.

    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ].

    Techniques: Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction

    Gel electrophoresis showing single base discretion using cTRACE. (A) Demonstration of cTRACE on targets within KIT exon 18 and TP53 exon 10 in normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment with and without adapter, respectively, and amplified with KIT exon 18 chimeric primers. Lanes 3 and 7 show the results of cTRACE amplification with two perfectly matched primers for the two targets. Lanes 4 and 8 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch at the 3’-end. Lanes 5 and 9 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch in the second base from the 3’-end. Lanes 6 and 10 show the results of cTRACE amplification with one perfectly matched primer and one primer with two mismatches in the first and second position from the 3’-end. (B) Demonstration of cTRACE on KRAS exon 2 in normal human genomic DNA. Lane 1 shows enrichment found with a perfect matched primer and Lane 2 shows enrichment found with a single mismatch on the 3’-end of the primer that matches the KRAS G12D mutation.

    Journal: PLoS ONE

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    doi: 10.1371/journal.pone.0243781

    Figure Lengend Snippet: Gel electrophoresis showing single base discretion using cTRACE. (A) Demonstration of cTRACE on targets within KIT exon 18 and TP53 exon 10 in normal human genomic DNA. Lanes 1 and 2 show controls without Cas9/sgRNA treatment with and without adapter, respectively, and amplified with KIT exon 18 chimeric primers. Lanes 3 and 7 show the results of cTRACE amplification with two perfectly matched primers for the two targets. Lanes 4 and 8 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch at the 3’-end. Lanes 5 and 9 show the results of cTRACE amplification with one perfectly matched primer and one primer with a single mismatch in the second base from the 3’-end. Lanes 6 and 10 show the results of cTRACE amplification with one perfectly matched primer and one primer with two mismatches in the first and second position from the 3’-end. (B) Demonstration of cTRACE on KRAS exon 2 in normal human genomic DNA. Lane 1 shows enrichment found with a perfect matched primer and Lane 2 shows enrichment found with a single mismatch on the 3’-end of the primer that matches the KRAS G12D mutation.

    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ].

    Techniques: Nucleic Acid Electrophoresis, Amplification, Mutagenesis

    Comparison of gel electrophoresis results for cCAMP and sequence specific PCR on five individual targets and a multiplex. (A) Gel electrophoresis results of cCAMP. Lanes 1 and 2 show controls without Cas9/sgRNA with and without UPS adapters; amplification is complete with UPS primers. Lanes 3–7 show the results of cCAMP targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 8 shows the five targets as a multiplex. Noted product lengths include 42 bp for the addition of the UPS adapters. (B) Gel results of sequence specific PCR designed to have the same melting temperature as the UPS component of the chimeric primers (63°C). (C) Gel results of sequence specific PCR designed to have the same melting temperature as the full chimeric primers (66°C). Sequence specific PCR were performed under same conditions as cCAMP (annealing temperature 65°C) and include a no template control in the first lane of each gel.

    Journal: PLoS ONE

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    doi: 10.1371/journal.pone.0243781

    Figure Lengend Snippet: Comparison of gel electrophoresis results for cCAMP and sequence specific PCR on five individual targets and a multiplex. (A) Gel electrophoresis results of cCAMP. Lanes 1 and 2 show controls without Cas9/sgRNA with and without UPS adapters; amplification is complete with UPS primers. Lanes 3–7 show the results of cCAMP targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 8 shows the five targets as a multiplex. Noted product lengths include 42 bp for the addition of the UPS adapters. (B) Gel results of sequence specific PCR designed to have the same melting temperature as the UPS component of the chimeric primers (63°C). (C) Gel results of sequence specific PCR designed to have the same melting temperature as the full chimeric primers (66°C). Sequence specific PCR were performed under same conditions as cCAMP (annealing temperature 65°C) and include a no template control in the first lane of each gel.

    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ].

    Techniques: Nucleic Acid Electrophoresis, Sequencing, Polymerase Chain Reaction, Multiplex Assay, Amplification

    cCAMP enrichment from a cfDNA model. (A) Gel electrophoresis of cCAMP with a cfDNA model input control reaction results. Lane 1 shows a control without Cas9/sgRNA, without UPS adapters, and is amplified with UPS primers. Lane 2 shows a control without Cas9/sgRNA with UPS adapter but no primers. Lane 3 shows a positive control without Cas9/sgRNA, with UPS adapters, and is amplified with a UPS primer. Lanes 4–8 show the results of the cCAMP protocol without the addition of Cas9/sgRNA complexes but with UPS adapters and chimeric primers for KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 9 shows the results of the multiplex without Cas9/sgRNA. (B) Gel electrophoresis of cCAMP with a cfDNA model input. Lanes 10–14 show the cCAMP procedure, including treatment with Cas9/sgRNA complexes targeting KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 15 shows results of the five targets as a multiplex. Note, the sizes listed only include the length required between the two Cas9/sgRNA complexes and do not include the length of the UPS adapter.

    Journal: PLoS ONE

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    doi: 10.1371/journal.pone.0243781

    Figure Lengend Snippet: cCAMP enrichment from a cfDNA model. (A) Gel electrophoresis of cCAMP with a cfDNA model input control reaction results. Lane 1 shows a control without Cas9/sgRNA, without UPS adapters, and is amplified with UPS primers. Lane 2 shows a control without Cas9/sgRNA with UPS adapter but no primers. Lane 3 shows a positive control without Cas9/sgRNA, with UPS adapters, and is amplified with a UPS primer. Lanes 4–8 show the results of the cCAMP protocol without the addition of Cas9/sgRNA complexes but with UPS adapters and chimeric primers for KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 9 shows the results of the multiplex without Cas9/sgRNA. (B) Gel electrophoresis of cCAMP with a cfDNA model input. Lanes 10–14 show the cCAMP procedure, including treatment with Cas9/sgRNA complexes targeting KIT exon 18, TP53 exon 4, CTNNB1 exon 4, NRAS exon 4, and TP53 exon 11, respectively. Lane 15 shows results of the five targets as a multiplex. Note, the sizes listed only include the length required between the two Cas9/sgRNA complexes and do not include the length of the UPS adapter.

    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ].

    Techniques: Nucleic Acid Electrophoresis, Amplification, Positive Control, Multiplex Assay

    Gel electrophoresis results of mutation specific enrichment by TRACE on a series of phosphorothioated PCR products. (A) TRACE on an 820 bp PCR product around the CFTR F2 sgRNA site with both primers phosphorothioated. (B) TRACE on the 820 bp PCR product with both primers phosphorylated. (C) TRACE on the 820 bp PCR product with the forward primer phosphorothioated and the reverse primer phosphorylated. (D) TRACE of the 820 bp PCR product with the forward primer phosphorylated and the reverse primer phosphorothioated. In each gel, (A)—(D), Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease. Lanes 3 and 4 show the results of Cas9/sgRNA treated reactions with a perfectly matched sgRNA without and with exonuclease treatment. Lanes 5 and 6 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the first position prior to the PAM site without and with exonuclease treatment. Lanes 7 and 8 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the third position prior to the PAM site without and with exonuclease treatment.

    Journal: PLoS ONE

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    doi: 10.1371/journal.pone.0243781

    Figure Lengend Snippet: Gel electrophoresis results of mutation specific enrichment by TRACE on a series of phosphorothioated PCR products. (A) TRACE on an 820 bp PCR product around the CFTR F2 sgRNA site with both primers phosphorothioated. (B) TRACE on the 820 bp PCR product with both primers phosphorylated. (C) TRACE on the 820 bp PCR product with the forward primer phosphorothioated and the reverse primer phosphorylated. (D) TRACE of the 820 bp PCR product with the forward primer phosphorylated and the reverse primer phosphorothioated. In each gel, (A)—(D), Lanes 1 and 2 show controls without Cas9/sgRNA treatment without and with exonuclease. Lanes 3 and 4 show the results of Cas9/sgRNA treated reactions with a perfectly matched sgRNA without and with exonuclease treatment. Lanes 5 and 6 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the first position prior to the PAM site without and with exonuclease treatment. Lanes 7 and 8 show the results of Cas9/sgRNA treated reactions with a mismatch in the sgRNA that produces a mismatch in the third position prior to the PAM site without and with exonuclease treatment.

    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ].

    Techniques: Nucleic Acid Electrophoresis, Mutagenesis, Polymerase Chain Reaction

    Evidence for Cas9/sgRNA produced staggered cut. (A) Gel electrophoresis results of cCAMP with dA-tailing only before ligation. Lane 1 shows a control reaction without Cas9 with UPS adapter and the TP53 chimeric primers. Lanes 2–6 show results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (B) Gel electrophoresis results of cCAMP with dA-tailing module and added dCTP, dGTP and dTTP. Lanes 0 and 1 show control reactions without Cas9/sgRNA with and without UPS adapters with amplification complete with UPS primers. Lanes 2–6 show cCAMP results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (C) Analysis of components for the DNA pre-ligation preparation and ligation for TP53 exon 10. (D) Analysis of components for the DNA pre-ligation preparation and ligation for MET exon 19. For (D) Lane 0 shows a control without Cas9 with UPS adapter and MET exon 19 primers with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation. For (B) and (D) Lane 1 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation, Lane 2 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and KAPA HyperPlus Ligation, Lane 3 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and KAPA HyperPlus Ligation, Lane 4 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and NEBNext Ultra II Ligation, Lane 5 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and NEB ligation, and Lane 6 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and NEBNext Ultra II Ligation. In all samples, Klenow (exo-) was included in pre-ligation reaction.

    Journal: PLoS ONE

    Article Title: Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations

    doi: 10.1371/journal.pone.0243781

    Figure Lengend Snippet: Evidence for Cas9/sgRNA produced staggered cut. (A) Gel electrophoresis results of cCAMP with dA-tailing only before ligation. Lane 1 shows a control reaction without Cas9 with UPS adapter and the TP53 chimeric primers. Lanes 2–6 show results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (B) Gel electrophoresis results of cCAMP with dA-tailing module and added dCTP, dGTP and dTTP. Lanes 0 and 1 show control reactions without Cas9/sgRNA with and without UPS adapters with amplification complete with UPS primers. Lanes 2–6 show cCAMP results targeting KIT exon 18, TP53 exon 10, MET exon 19, GNAQ exon 5, and PDGFRA exon 18, respectively. Lane 7 shows the five targets as a multiplex. (C) Analysis of components for the DNA pre-ligation preparation and ligation for TP53 exon 10. (D) Analysis of components for the DNA pre-ligation preparation and ligation for MET exon 19. For (D) Lane 0 shows a control without Cas9 with UPS adapter and MET exon 19 primers with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation. For (B) and (D) Lane 1 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and KAPA HyperPlus Ligation, Lane 2 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and KAPA HyperPlus Ligation, Lane 3 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and KAPA HyperPlus Ligation, Lane 4 shows cCAMP with NEBNext Ultra II dA-tailing (dATP) and NEBNext Ultra II Ligation, Lane 5 shows cCAMP with NEBNext Ultra II dA-tailing with three additional dNTPs added (four dNTPs) and NEB ligation, and Lane 6 shows cCAMP with KAPA HyperPlus End Repair A-Tailing Buffer (four dNTPs) and NEBNext Ultra II Ligation. In all samples, Klenow (exo-) was included in pre-ligation reaction.

    Article Snippet: Recently, others have reported that the endonuclease activity of Cas9 produces staggered ends, contrary to the previous convention that Cas9/sgRNA produces blunt-end cuts [ – ].

    Techniques: Produced, Nucleic Acid Electrophoresis, Ligation, Multiplex Assay, Amplification

    Domain insertion into AcrIIC1 yields a highly potent Nme Cas9 inhibitor. ( a , b ) HEK 293T cells were co-transfected with vectors expressing Nme Cas9, the indicated Acr and sgRNAs targeting different genomic loci followed by T7 endonuclease assay. In a , Acr: Nme Cas9 vector ratio used during transfection was 1:1, while in b , the indicated, low Acr: Nme Cas9 vector ratios were used. Representative T7 gel images and corresponding quantification of indel frequencies are shown. Lines in plots show means, dots are individual data points for n = 4 (DHFR, AAVS1 and IL2RG locus) or n = 3 (F8 locus) independent experiments. Chim., AcrIIC1-mCherry chimeras in Supplementary Fig. 2. In., input band. T7, T7 cleavage fragments. ( c ) AcrIIC1-mCherry chimeras outperform wild-type AcrIIC1 and AcrIIC3. Cells were co-transfected with vectors encoding Nme Cas9, a firefly luciferase reporter and corresponding reporter-targeting sgRNA as well as the indicated Acr followed by luciferase assay. Bars indicate means, error bars the SD for n = 3 independent experiments. ( a-c ) N, negative (Cas 9 only ( a , b ) or reporter only ( c ) control (Ctrl.)). P, positive control (Cas9 + sgRNA ( a , b ) or reporter + Cas9 ( c )). * P

    Journal: bioRxiv

    Article Title: Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity

    doi: 10.1101/685032

    Figure Lengend Snippet: Domain insertion into AcrIIC1 yields a highly potent Nme Cas9 inhibitor. ( a , b ) HEK 293T cells were co-transfected with vectors expressing Nme Cas9, the indicated Acr and sgRNAs targeting different genomic loci followed by T7 endonuclease assay. In a , Acr: Nme Cas9 vector ratio used during transfection was 1:1, while in b , the indicated, low Acr: Nme Cas9 vector ratios were used. Representative T7 gel images and corresponding quantification of indel frequencies are shown. Lines in plots show means, dots are individual data points for n = 4 (DHFR, AAVS1 and IL2RG locus) or n = 3 (F8 locus) independent experiments. Chim., AcrIIC1-mCherry chimeras in Supplementary Fig. 2. In., input band. T7, T7 cleavage fragments. ( c ) AcrIIC1-mCherry chimeras outperform wild-type AcrIIC1 and AcrIIC3. Cells were co-transfected with vectors encoding Nme Cas9, a firefly luciferase reporter and corresponding reporter-targeting sgRNA as well as the indicated Acr followed by luciferase assay. Bars indicate means, error bars the SD for n = 3 independent experiments. ( a-c ) N, negative (Cas 9 only ( a , b ) or reporter only ( c ) control (Ctrl.)). P, positive control (Cas9 + sgRNA ( a , b ) or reporter + Cas9 ( c )). * P

    Article Snippet: Transfections for the initial screen of the chimeric AcrIIC1 variants (Supplementary Fig. 2) were performed with only 100 ng of total DNA per well, using a 1:1 ratio of Cas9/sgRNA and Acr vectors.

    Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Positive Control

    PGRMC1 knockout using a CRISPR/Cas9 approach. A. Schematic of the PGRMC1 sgRNA/Cas9-expressing lentiviral constructs for knocking out PGRMC1. Gray areas show the three candidate sgRNA sequences in the exon-1 of the PGRMC1 gene, of which two were used for the CRISPR/Cas9 constructs. B. A representative DNA gel of control and PGRMC1 knockout (clones 38 and 207) NSC34 cells verifying the Cas9 cleavage of the genomic DNA. Control refers to the NSC34 cells transfected with the control vector expressing Cas9 but not an sgRNA. C. Western blotting detection of PGRMC1 in control and PGRMC1 knockout (clones 38 and 207) NSC34 cells.

    Journal: EBioMedicine

    Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

    doi: 10.1016/j.ebiom.2015.10.017

    Figure Lengend Snippet: PGRMC1 knockout using a CRISPR/Cas9 approach. A. Schematic of the PGRMC1 sgRNA/Cas9-expressing lentiviral constructs for knocking out PGRMC1. Gray areas show the three candidate sgRNA sequences in the exon-1 of the PGRMC1 gene, of which two were used for the CRISPR/Cas9 constructs. B. A representative DNA gel of control and PGRMC1 knockout (clones 38 and 207) NSC34 cells verifying the Cas9 cleavage of the genomic DNA. Control refers to the NSC34 cells transfected with the control vector expressing Cas9 but not an sgRNA. C. Western blotting detection of PGRMC1 in control and PGRMC1 knockout (clones 38 and 207) NSC34 cells.

    Article Snippet: In order to assess the efficiency of sgRNA-guided Cas9 cutting in the PGRMC1 genomic sequence (exon-1), genomic DNA was extracted for PCR amplification of the specific region including the sgRNA/Cas9 excision site.

    Techniques: Knock-Out, CRISPR, Expressing, Construct, Transfection, Plasmid Preparation, Western Blot

    Eliminating PGRMC1 protein does not alter [ 3 H]-DTG binding to the S2R in cell membranes. A. A representative of [ 3 H]-DTG saturation binding in membranes prepared from control and PGRMC1 knockout (clones 38 and 207) NSC34 cells, (+)-pentazocine (100 nM) was included to mask [ 3 H]-DTG binding to the S1R such that [ 3 H]-DTG would be bound only to the S2R and measured as specific S2R binding. Nonspecific binding was measured (by adding haloperidol) and subtracted. Control refers to the NSC34 cells transfected with the control vector for the expression of Cas9 but not an sgRNA. B. Statistics. Maximum binding (B max ) and equilibrium dissociation constants (K D ) for [ 3 H]-DTG were calculated using a Prizm software and reported as mean ± SEM of three separate experiments each performed in triplicates. Not significant (n.s.).

    Journal: EBioMedicine

    Article Title: The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

    doi: 10.1016/j.ebiom.2015.10.017

    Figure Lengend Snippet: Eliminating PGRMC1 protein does not alter [ 3 H]-DTG binding to the S2R in cell membranes. A. A representative of [ 3 H]-DTG saturation binding in membranes prepared from control and PGRMC1 knockout (clones 38 and 207) NSC34 cells, (+)-pentazocine (100 nM) was included to mask [ 3 H]-DTG binding to the S1R such that [ 3 H]-DTG would be bound only to the S2R and measured as specific S2R binding. Nonspecific binding was measured (by adding haloperidol) and subtracted. Control refers to the NSC34 cells transfected with the control vector for the expression of Cas9 but not an sgRNA. B. Statistics. Maximum binding (B max ) and equilibrium dissociation constants (K D ) for [ 3 H]-DTG were calculated using a Prizm software and reported as mean ± SEM of three separate experiments each performed in triplicates. Not significant (n.s.).

    Article Snippet: In order to assess the efficiency of sgRNA-guided Cas9 cutting in the PGRMC1 genomic sequence (exon-1), genomic DNA was extracted for PCR amplification of the specific region including the sgRNA/Cas9 excision site.

    Techniques: Binding Assay, Knock-Out, Transfection, Plasmid Preparation, Expressing, Software