engen sgrna synthesis kit  (New England Biolabs)


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    Name:
    EnGen sgRNA Synthesis Kit S pyogenes
    Description:
    EnGen sgRNA Synthesis Kit S pyogenes 20 rxns
    Catalog Number:
    E3322S
    Price:
    416
    Category:
    Transcription Kits
    Size:
    20 rxns
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    New England Biolabs engen sgrna synthesis kit
    EnGen sgRNA Synthesis Kit S pyogenes
    EnGen sgRNA Synthesis Kit S pyogenes 20 rxns
    https://www.bioz.com/result/engen sgrna synthesis kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    engen sgrna synthesis kit - by Bioz Stars, 2021-06
    99/100 stars

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    other:

    Article Title: Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes
    Article Snippet: For different ratios of sgRNA/Cas9 and peptides, we found complexes of ranging sizes (mean±SE) from 88.4±1.9 nm to 115.3±6.0 nm ( and ).

    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. ).

    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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  • 99
    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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    Average 99 stars, based on 1 article reviews
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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

    Identification of heterozygous F1 animals with CRISPR/Cas9 indel mutations of the mtnr1a gene. ( a ) Representative RFLP analysis shows that PCR products of mtnr1a VIL heterozygotes display two predicted cleavage bands (arrows), whereas WT and non-VIL mutant DNA is not cleaved. The creation of a BseRI site by the VIL deletion is illustrated in Supplementary Fig. S3 . ( b ) Direct PCR Sanger sequencing chromatogram of F1 WT progeny display prominent peaks for all bases in the T2 sgRNA target area. The T2 PAM sequence (CCT) is boxed in black, and the downstream 9-bp sequence of the mtnr1a VIL deletion mutation is boxed in blue. The corresponding amino acid sequence is shown below the nucleotide codon sequence. ( c ) In an F1 tadpole heterozygous for the mtnr1a 9-bp (VIL) deletion, the trace decomposes (i.e., mixed base calls) at the predicted target site 4-bp downstream of the T2 PAM site, and continues to the end of the trace, as expected for a heterozygous indel mutation. The decomposed sequence represents the overlapping peaks of the mtnr1a WT and VIL alleles. ( d ) Partial view of alignments from Poly Peak Parser ( https://yosttools.genetics.utah.edu/PolyPeakParser/ ) output revealing the heterozygous 9-bp mtnr1a VIL deletion mutation. The WT allele is the top line and the mutant allele is the bottom line.

    Journal: Scientific Reports

    Article Title: CRISPR/Cas9 mediated mutation of the mtnr1a melatonin receptor gene causes rod photoreceptor degeneration in developing Xenopus tropicalis

    doi: 10.1038/s41598-020-70735-2

    Figure Lengend Snippet: Identification of heterozygous F1 animals with CRISPR/Cas9 indel mutations of the mtnr1a gene. ( a ) Representative RFLP analysis shows that PCR products of mtnr1a VIL heterozygotes display two predicted cleavage bands (arrows), whereas WT and non-VIL mutant DNA is not cleaved. The creation of a BseRI site by the VIL deletion is illustrated in Supplementary Fig. S3 . ( b ) Direct PCR Sanger sequencing chromatogram of F1 WT progeny display prominent peaks for all bases in the T2 sgRNA target area. The T2 PAM sequence (CCT) is boxed in black, and the downstream 9-bp sequence of the mtnr1a VIL deletion mutation is boxed in blue. The corresponding amino acid sequence is shown below the nucleotide codon sequence. ( c ) In an F1 tadpole heterozygous for the mtnr1a 9-bp (VIL) deletion, the trace decomposes (i.e., mixed base calls) at the predicted target site 4-bp downstream of the T2 PAM site, and continues to the end of the trace, as expected for a heterozygous indel mutation. The decomposed sequence represents the overlapping peaks of the mtnr1a WT and VIL alleles. ( d ) Partial view of alignments from Poly Peak Parser ( https://yosttools.genetics.utah.edu/PolyPeakParser/ ) output revealing the heterozygous 9-bp mtnr1a VIL deletion mutation. The WT allele is the top line and the mutant allele is the bottom line.

    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. ) .

    Techniques: CRISPR, Polymerase Chain Reaction, Mutagenesis, Sequencing

    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