cas12a  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    EnGen Lba Cas12a Cpf1
    Description:
    EnGen Lba Cas12a Cpf1 70 pmol
    Catalog Number:
    M0653S
    Price:
    70
    Category:
    Other Endonucleases
    Size:
    70 pmol
    Buy from Supplier


    Structured Review

    New England Biolabs cas12a
    EnGen Lba Cas12a Cpf1
    EnGen Lba Cas12a Cpf1 70 pmol
    https://www.bioz.com/result/cas12a/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas12a - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) Product Images from "Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles"

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa020

    CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.
    Figure Legend Snippet: CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.

    Techniques Used: Incubation

    CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).
    Figure Legend Snippet: CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).

    Techniques Used: Fluorescence, Activation Assay

    2) Product Images from "A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020"

    Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.02.012

    PAM usage of Lb, Lb2, and Lb2-KY (A) A diagram outlining the mammalian-cell PAM identification assay used in subsequent panels. A library of randomized 5′-NNNNNN-3′ PAMs preceding a crRNA target was stably expressed in HEK293T cells. Plasmids encoding Cas12a proteins, crRNAs, and the fluorescent marker mCherry were transfected to these cells. Cells were sorted for mCherry expression, and a region containing the randomized PAM and the target sequence was deep-sequenced with paired-end reads of 300 base pairs. Indel frequencies were then calculated for each four-nucleotide PAM. Note that the first two nucleotides of the 6-mer randomized PAM sequence had no detectable impact on the activities of any Cas12a variant. (B) Heatmaps showing the result of mammalian-cell PAM screen for Lb2Cas12a, Lb2-KY, LbCas12a, and cells lacking a Cas12a ortholog (control). PAMs and the related editing efficiencies were pooled by the first three nucleotide positions. Each heatmap indicates an average of two or three independent replicates. The range of replicates for each 3-nucleotide PAM sequence is less than 10% of the signal at each position. (C) An expansion of the data generated in (B), except that all 64 four-nucleotide PAMs are plotted. The range of replicates for each 4-nucleotide sequence is less than 25% of the average signal for each four-nucleotide PAM.
    Figure Legend Snippet: PAM usage of Lb, Lb2, and Lb2-KY (A) A diagram outlining the mammalian-cell PAM identification assay used in subsequent panels. A library of randomized 5′-NNNNNN-3′ PAMs preceding a crRNA target was stably expressed in HEK293T cells. Plasmids encoding Cas12a proteins, crRNAs, and the fluorescent marker mCherry were transfected to these cells. Cells were sorted for mCherry expression, and a region containing the randomized PAM and the target sequence was deep-sequenced with paired-end reads of 300 base pairs. Indel frequencies were then calculated for each four-nucleotide PAM. Note that the first two nucleotides of the 6-mer randomized PAM sequence had no detectable impact on the activities of any Cas12a variant. (B) Heatmaps showing the result of mammalian-cell PAM screen for Lb2Cas12a, Lb2-KY, LbCas12a, and cells lacking a Cas12a ortholog (control). PAMs and the related editing efficiencies were pooled by the first three nucleotide positions. Each heatmap indicates an average of two or three independent replicates. The range of replicates for each 3-nucleotide PAM sequence is less than 10% of the signal at each position. (C) An expansion of the data generated in (B), except that all 64 four-nucleotide PAMs are plotted. The range of replicates for each 4-nucleotide sequence is less than 25% of the average signal for each four-nucleotide PAM.

    Techniques Used: Stable Transfection, Marker, Transfection, Expressing, Sequencing, Variant Assay, Generated

    Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. 21 , 22 (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. 11 Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of AsCas12a, LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p
    Figure Legend Snippet: Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. 21 , 22 (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. 11 Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of AsCas12a, LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p

    Techniques Used: Generated, Binding Assay, Expressing, Construct, Sequencing, Luciferase, Stable Transfection, Non-Homologous End Joining, Activity Assay

    3) Product Images from "gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing"

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.110494

    Comparison of Cas9 and Cas12a editing efficiency with T7RNAP variants. Efficiency of ADE2 editing by Cas12- or Cas9-mediated gEL DNA in T7RNAP mutant or overexpression strains: IMX1905 (K276R); IMX2031 (wild-type, wt ); IMX2032 (P266L); IMX2030 (P266L_K276R); IME459 (K276R overexpression, ↗K276R); IME475 (P266L overexpression, P266L). For Cas12a, transformed gDNA corresponds to annealed 15093-15094 oligos. For Cas9, transformed gDNA was obtained by PCR-derived fragment using overlapping primers 16745-16746. Editing efficiency is expressed as percentage of red colonies ( ade2 − ). Values represent the average and standard deviations of data obtained from independent biological duplicates. * P
    Figure Legend Snippet: Comparison of Cas9 and Cas12a editing efficiency with T7RNAP variants. Efficiency of ADE2 editing by Cas12- or Cas9-mediated gEL DNA in T7RNAP mutant or overexpression strains: IMX1905 (K276R); IMX2031 (wild-type, wt ); IMX2032 (P266L); IMX2030 (P266L_K276R); IME459 (K276R overexpression, ↗K276R); IME475 (P266L overexpression, P266L). For Cas12a, transformed gDNA corresponds to annealed 15093-15094 oligos. For Cas9, transformed gDNA was obtained by PCR-derived fragment using overlapping primers 16745-16746. Editing efficiency is expressed as percentage of red colonies ( ade2 − ). Values represent the average and standard deviations of data obtained from independent biological duplicates. * P

    Techniques Used: Mutagenesis, Over Expression, Transformation Assay, Polymerase Chain Reaction, Derivative Assay

    Optimization of Cas9 and Cas12a gDNA design. Editing efficiency of ADE2 in strain IMX1905 transformed with gDNAs for cloning-free, T7RNAP-driven expression of gRNA. A) gDNA configurations for Cas9-mediated genome editing and respective editing efficiencies. B) gDNA configurations for Cas12a-mediated genome editing and their respective editing efficiencies. The size of each gDNA is specified on the right of the respective graph bar. Editing efficiency is expressed as percentage of red colonies ( ade2 ) over the total number of colonies. Values represent the average and standard deviations of data obtained from three independent biological replicates.
    Figure Legend Snippet: Optimization of Cas9 and Cas12a gDNA design. Editing efficiency of ADE2 in strain IMX1905 transformed with gDNAs for cloning-free, T7RNAP-driven expression of gRNA. A) gDNA configurations for Cas9-mediated genome editing and respective editing efficiencies. B) gDNA configurations for Cas12a-mediated genome editing and their respective editing efficiencies. The size of each gDNA is specified on the right of the respective graph bar. Editing efficiency is expressed as percentage of red colonies ( ade2 ) over the total number of colonies. Values represent the average and standard deviations of data obtained from three independent biological replicates.

    Techniques Used: Transformation Assay, Clone Assay, Expressing

    Multiplex genome editing by Cas12a-mediated using the gEL DNA approach. (A) Targeted sites for deletion of ADE2 (ADE2-3, green), HIS4 (HIS4-4, orange), PDR12 (PDR12-3, cyan) and CAN1 (CAN1-4, pink; CAN1-3, violet) genes. (B) Percentage of transformants obtained from double gDNAs delivery: ADE2-3 and HIS4-4. (C) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-4. (D) Verification of single editing efficiency of CAN1 targets expressed from plasmid pUDR717 (CAN1-4) or pUDR718 (CAN1-3), with prediction of gRNAs secondary structure. (E) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-3. Number of verified clones is indicated between brackets and diagnostic PCRs are reported in Supplementary Figures (Fig. S4, S5, S6). Zero (0Δ), single (1Δ), double (2Δ), triple (3Δ) or quadruple (4Δ) deletion are indicated at the outside ends of each fraction. Type of obtained deletions are specified with the respective colour of the target. Number of colonies are also stated next to each depiction. Prediction of the gRNA stem-loop for Cas12a recognition is highlighted by a red square.
    Figure Legend Snippet: Multiplex genome editing by Cas12a-mediated using the gEL DNA approach. (A) Targeted sites for deletion of ADE2 (ADE2-3, green), HIS4 (HIS4-4, orange), PDR12 (PDR12-3, cyan) and CAN1 (CAN1-4, pink; CAN1-3, violet) genes. (B) Percentage of transformants obtained from double gDNAs delivery: ADE2-3 and HIS4-4. (C) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-4. (D) Verification of single editing efficiency of CAN1 targets expressed from plasmid pUDR717 (CAN1-4) or pUDR718 (CAN1-3), with prediction of gRNAs secondary structure. (E) Fraction of selected colonies upon transformation with four gDNAs: ADE2-3, HIS4-4, PDR12-3 and CAN1-3. Number of verified clones is indicated between brackets and diagnostic PCRs are reported in Supplementary Figures (Fig. S4, S5, S6). Zero (0Δ), single (1Δ), double (2Δ), triple (3Δ) or quadruple (4Δ) deletion are indicated at the outside ends of each fraction. Type of obtained deletions are specified with the respective colour of the target. Number of colonies are also stated next to each depiction. Prediction of the gRNA stem-loop for Cas12a recognition is highlighted by a red square.

    Techniques Used: Multiplex Assay, Transformation Assay, Plasmid Preparation, Clone Assay, Diagnostic Assay

    Physiological characterization of S. cerevisiae strains expressing T7RNAP. Maximum specific growth rates ( μ max ) of S. cerevisiae constitutively expressing T7RNAP K276R , Cas9 and Cas12a (IMX1905) and its control strain (CENPK.113-7D), or S. cerevisiae strains overexpressing T7RNAP K276R (IME459) or T7RNAP P266L (IME475) and its control strain carrying a 2μm multi-copy empty vector (IME460). Strains were cultivated in 96-well plate containing chemically defined medium supplemented with glucose as sole carbon source. Data points represent average and mean deviations of four biological replicates. *P
    Figure Legend Snippet: Physiological characterization of S. cerevisiae strains expressing T7RNAP. Maximum specific growth rates ( μ max ) of S. cerevisiae constitutively expressing T7RNAP K276R , Cas9 and Cas12a (IMX1905) and its control strain (CENPK.113-7D), or S. cerevisiae strains overexpressing T7RNAP K276R (IME459) or T7RNAP P266L (IME475) and its control strain carrying a 2μm multi-copy empty vector (IME460). Strains were cultivated in 96-well plate containing chemically defined medium supplemented with glucose as sole carbon source. Data points represent average and mean deviations of four biological replicates. *P

    Techniques Used: Expressing, Plasmid Preparation

    Schematic overview of the gEL DNA approach. 1, in silico design and ordering of gDNA cassettes (87 bp) and repair DNA (120 bp) as oligos. 2, tranformation with the double-stranded (ds) gDNA expression cassettes (2a), the ds repair DNA fragments and an empty, split plasmid carrying a marker of choice. 3, expression of the gRNA by the T7RNAP. 4, targetted DNA editing by Cas12a (Cas12a). 5, repair of the ds DNA break via homologous recombination using the repair DNA fragments.
    Figure Legend Snippet: Schematic overview of the gEL DNA approach. 1, in silico design and ordering of gDNA cassettes (87 bp) and repair DNA (120 bp) as oligos. 2, tranformation with the double-stranded (ds) gDNA expression cassettes (2a), the ds repair DNA fragments and an empty, split plasmid carrying a marker of choice. 3, expression of the gRNA by the T7RNAP. 4, targetted DNA editing by Cas12a (Cas12a). 5, repair of the ds DNA break via homologous recombination using the repair DNA fragments.

    Techniques Used: In Silico, Expressing, Plasmid Preparation, Marker, Homologous Recombination

    Related Articles

    other:

    Article Title: Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA
    Article Snippet: For each target, two Cas9-crRNA were designed to flank the region of interest to be protected and two Cas12a-crRNA at least 100 bases away from the Cas9-crRNA position. (The complete list of crRNA is available in Supplementary Data ).

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles
    Article Snippet: Fortuitously, all three key components in the reaction, Cas12a, TdT and CuNPs, function optimally in weakly alkaline conditions.

    In Vitro:

    Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020
    Article Snippet: At the DNMT1-3 target, Lb2-KY activity was slightly less than that of As-Ultra. .. The trans-cleavage and off-target effects of Lb2-KY Cas12a nucleases have been shown to indiscriminately cleave ssDNA in vitro after being activated by a bona fide double-stranded DNA target., We used ssDNA substrates with fluorophore-quencher to study the trans-cleavage activity of Lb2-KY, as previously described., Lb2-KY showed comparable trans-cleavage activity to LbCas12a (i.e., EnGen Lba Cas12a, NEB); both were activated by targets containing TTTV and CTTC PAMs, but not AGCG or ACTG targets ( A–S7D). .. Note that it is not clear that this trans-cleavage activity is deleterious in physiological settings, because ssDNA in replication bubbles and homologous recombination complexes are protected by DNA-binding proteins, , , and because LbCas12a-mediated HDR with ssDNA repair templates is as efficient as that mediated by AsCas12a.

    Activity Assay:

    Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020
    Article Snippet: At the DNMT1-3 target, Lb2-KY activity was slightly less than that of As-Ultra. .. The trans-cleavage and off-target effects of Lb2-KY Cas12a nucleases have been shown to indiscriminately cleave ssDNA in vitro after being activated by a bona fide double-stranded DNA target., We used ssDNA substrates with fluorophore-quencher to study the trans-cleavage activity of Lb2-KY, as previously described., Lb2-KY showed comparable trans-cleavage activity to LbCas12a (i.e., EnGen Lba Cas12a, NEB); both were activated by targets containing TTTV and CTTC PAMs, but not AGCG or ACTG targets ( A–S7D). .. Note that it is not clear that this trans-cleavage activity is deleterious in physiological settings, because ssDNA in replication bubbles and homologous recombination complexes are protected by DNA-binding proteins, , , and because LbCas12a-mediated HDR with ssDNA repair templates is as efficient as that mediated by AsCas12a.

    Concentration Assay:

    Article Title: Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology
    Article Snippet: Cas12a-mediated transcleavage of the ssDNA reporter, for the purpose of fluorescence detection, was carried out at room temperature (approximately 23 °C) for 10 min. .. The EnGen Lba Cas12a enzyme (NEB) at 1 μM concentration was preincubated with 1.25 μM gRNA in 1× NEBuffer 2.1 (NEB) to form the ribonucleoprotein (RNP) complex. .. An aliquot of the RNP complex solution was placed inside the cap of a PCR tube.

    CRISPR:

    Article Title: Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens
    Article Snippet: Targeted genes of SARS-CoV-2 were amplified by incubating at 39 °C for 30 min, then the reaction was inactivated at 75 °C for 5 min. .. The CRISPR-Cas12a detection method consists of 1X NEBuffer 2.0 (New England Biolabs, USA), 30 nM crRNA, 330 nM EnGen® Lba Cas12a endonuclease (New England Biolabs, USA), 200 nM 5' 6-FAM / 3' BHQ-1®, Dual Labeled Fluorescent Probe and DEPC-treated water in a total reaction volume of 15 µl. .. The amplified products were added in a CRISPR-Cas12a reaction and then incubated at 39 °C for 15 min.

    Article Title: Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus
    Article Snippet: Finally, crRNAs were quantified using the Nanodrop 2000C (Thermo Fisher Scientific) and stored at − 80 °C. .. CRISPR-Cas12a assay The detection of ASFV by probe-based qPCR has been described previously [ – ]. .. In terms of African swine fever virus (ASFV), qPCR is the gold standard, which we used as a reference for our CRISPR-Cas12a assay.

    Labeling:

    Article Title: Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens
    Article Snippet: Targeted genes of SARS-CoV-2 were amplified by incubating at 39 °C for 30 min, then the reaction was inactivated at 75 °C for 5 min. .. The CRISPR-Cas12a detection method consists of 1X NEBuffer 2.0 (New England Biolabs, USA), 30 nM crRNA, 330 nM EnGen® Lba Cas12a endonuclease (New England Biolabs, USA), 200 nM 5' 6-FAM / 3' BHQ-1®, Dual Labeled Fluorescent Probe and DEPC-treated water in a total reaction volume of 15 µl. .. The amplified products were added in a CRISPR-Cas12a reaction and then incubated at 39 °C for 15 min.

    Expressing:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Plasmid Preparation:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Polymerase Chain Reaction:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Amplification:

    Article Title: gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing
    Article Snippet: The backbone for assembly of Cas12a-gRNAs with SNR52p/SUP4t flanks was obtained by PCR amplification with primers 12710-5793. .. For T7RNAP-mediated expression of gRNAs via Cas12a, plasmid backbone was obtained by PCR amplification using either primers 14274-13713 (S.T7p/T7t ) or 14275-13713 (L.T7p/T7t ). .. Plasmid pUDE759 was assembled by SNR52p/SUP4t backbone fragment with annealed oligos 12713-12714.

    Real-time Polymerase Chain Reaction:

    Article Title: Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus
    Article Snippet: Finally, crRNAs were quantified using the Nanodrop 2000C (Thermo Fisher Scientific) and stored at − 80 °C. .. CRISPR-Cas12a assay The detection of ASFV by probe-based qPCR has been described previously [ – ]. .. In terms of African swine fever virus (ASFV), qPCR is the gold standard, which we used as a reference for our CRISPR-Cas12a assay.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs cas12a
    CANTRIP can be performed in a two-step reaction without <t>Cas12a</t> heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.
    Cas12a, 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
    https://www.bioz.com/result/cas12a/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas12a - by Bioz Stars, 2021-04
    99/100 stars
      Buy from Supplier

    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
    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, 2021-04
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs cas9 protein
    Laminins perform multiple roles during zebrafish heart morphogenesis A-D: mRNA in situ hybridization analysis of myl7 expression in control embryos injected with lamc1 -targeting gRNAs only (A,C) or with lamc1 -targeting gRNAs together with <t>Cas9</t> protein ( Lamc1 F0 , B-D) at 55hpf and 72hpf. E-H: Quantitative analysis of looping ratio (E, G) and myl7 area (F,H) in gRNA-injected controls (55hpf: n=44; 72hpf: n=44) and lamc1 F0 crispants (55hpf: n=47; 72hpf: n=44). lamc1 crispants exhibit reduced heart looping at 55hpf and 72hpf, and an increased area of myl7 expression at 72hpf. Horizontal bars indicate median with interquartile range, comparative statistics performed using Kruskal-Wallis test. I-L: mRNA in situ hybridization analysis of myl7 expression in sibling (I,K) and lamb1a Δ25 mutants (J,L) at 55hpf and 72hpf. M-P: Quantitative analysis of looping ratio (M,O) and myl7 area (N,P) in sibling (55hpf: n=70; 72hpf: n=56) and lamb1a Δ25 mutant embryos (55hpf: n=25; 72hpf: n=34). lamb1a Δ25 mutants exhibit a mild reduction in heart looping at 55hpf, and an increased area of myl7 expression at 55hpf and 72hpf. Scale bars: 50μm. Comparative statistics performed using Mann Whitney test, **** = p
    Cas9 Protein, 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
    https://www.bioz.com/result/cas9 protein/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cas9 protein - by Bioz Stars, 2021-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.

    Journal: Biology Methods & Protocols

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles

    doi: 10.1093/biomethods/bpaa020

    Figure Lengend Snippet: CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.

    Article Snippet: Fortuitously, all three key components in the reaction, Cas12a, TdT and CuNPs, function optimally in weakly alkaline conditions.

    Techniques: Incubation

    CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).

    Journal: Biology Methods & Protocols

    Article Title: Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles

    doi: 10.1093/biomethods/bpaa020

    Figure Lengend Snippet: CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).

    Article Snippet: Fortuitously, all three key components in the reaction, Cas12a, TdT and CuNPs, function optimally in weakly alkaline conditions.

    Techniques: Fluorescence, Activation Assay

    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

    Laminins perform multiple roles during zebrafish heart morphogenesis A-D: mRNA in situ hybridization analysis of myl7 expression in control embryos injected with lamc1 -targeting gRNAs only (A,C) or with lamc1 -targeting gRNAs together with Cas9 protein ( Lamc1 F0 , B-D) at 55hpf and 72hpf. E-H: Quantitative analysis of looping ratio (E, G) and myl7 area (F,H) in gRNA-injected controls (55hpf: n=44; 72hpf: n=44) and lamc1 F0 crispants (55hpf: n=47; 72hpf: n=44). lamc1 crispants exhibit reduced heart looping at 55hpf and 72hpf, and an increased area of myl7 expression at 72hpf. Horizontal bars indicate median with interquartile range, comparative statistics performed using Kruskal-Wallis test. I-L: mRNA in situ hybridization analysis of myl7 expression in sibling (I,K) and lamb1a Δ25 mutants (J,L) at 55hpf and 72hpf. M-P: Quantitative analysis of looping ratio (M,O) and myl7 area (N,P) in sibling (55hpf: n=70; 72hpf: n=56) and lamb1a Δ25 mutant embryos (55hpf: n=25; 72hpf: n=34). lamb1a Δ25 mutants exhibit a mild reduction in heart looping at 55hpf, and an increased area of myl7 expression at 55hpf and 72hpf. Scale bars: 50μm. Comparative statistics performed using Mann Whitney test, **** = p

    Journal: bioRxiv

    Article Title: Lamb1a regulates atrial growth by limiting excessive, contractility-dependent second heart field addition during zebrafish heart development

    doi: 10.1101/2021.03.10.434727

    Figure Lengend Snippet: Laminins perform multiple roles during zebrafish heart morphogenesis A-D: mRNA in situ hybridization analysis of myl7 expression in control embryos injected with lamc1 -targeting gRNAs only (A,C) or with lamc1 -targeting gRNAs together with Cas9 protein ( Lamc1 F0 , B-D) at 55hpf and 72hpf. E-H: Quantitative analysis of looping ratio (E, G) and myl7 area (F,H) in gRNA-injected controls (55hpf: n=44; 72hpf: n=44) and lamc1 F0 crispants (55hpf: n=47; 72hpf: n=44). lamc1 crispants exhibit reduced heart looping at 55hpf and 72hpf, and an increased area of myl7 expression at 72hpf. Horizontal bars indicate median with interquartile range, comparative statistics performed using Kruskal-Wallis test. I-L: mRNA in situ hybridization analysis of myl7 expression in sibling (I,K) and lamb1a Δ25 mutants (J,L) at 55hpf and 72hpf. M-P: Quantitative analysis of looping ratio (M,O) and myl7 area (N,P) in sibling (55hpf: n=70; 72hpf: n=56) and lamb1a Δ25 mutant embryos (55hpf: n=25; 72hpf: n=34). lamb1a Δ25 mutants exhibit a mild reduction in heart looping at 55hpf, and an increased area of myl7 expression at 55hpf and 72hpf. Scale bars: 50μm. Comparative statistics performed using Mann Whitney test, **** = p

    Article Snippet: To generate lamb1a (ENSDART00000170673.2) mutant zebrafish, a single gRNA targeting Exon 6 (5’-GGATCCTCAATCCTGAAGGCAGG-3’) was injected together with Cas9 protein and Phenol Red into the yolk at the 1-cell stage.

    Techniques: In Situ Hybridization, Expressing, Injection, Mutagenesis, MANN-WHITNEY