cas12a (New England Biolabs)
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Name:
EnGen Lba Cas12a Cpf1
Description:
EnGen Lba Cas12a Cpf1 70 pmol
Catalog Number:
M0653S
Price:
70
Category:
Other Endonucleases
Size:
70 pmol
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Structured Review
New England Biolabs
cas12a
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
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
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
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
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
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
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
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
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
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
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 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 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 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 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. .. 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 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 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 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 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 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. .. |
