Engen lba cas12a cpf1

Manufactured by New England Biolabs

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EnGen® Lba Cas12a (Cpf1) is a programmable RNA-guided DNA endonuclease derived from Lachnospiraceae bacterium. It recognizes a T-rich protospacer adjacent motif (PAM) and cleaves double-stranded DNA in a highly specific manner.

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EnGen® Lba Cas12a (28 citations) Lba Cas12a (19 citations) Cas12a (16 citations)

17 protocols using engen lba cas12a cpf1

CRISPR/Cas12a-mediated Deletion of foxi3a Promoter

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CRISPR/Cas12a-mediated gene editing was performed to delete the foxi3a promoter and a part of the first exon (Moreno-Mateos et al., 2017 (link)). Two guide RNAs (gRNAs) targeting sequences 1377 bp upstream and 143 bp downstream of the start codon, respectively, were designed using DeepCpf1 (Luo et al., 2019 (link)) and checked for specificity and off-target effects by zebrafish genome BLAST (Altschul et al., 1990 (link)) and CRISPRscan (Moreno-Mateos et al., 2017 (link)): gRNA 1: 5’-ATATGACAACCTACCGCAGA-3’; gRNA2: 5’-GGAGAATACGCAGGGCAGAC-3’.
Tg(tp1bglobin:EGFP)^um13 zebrafish one cell stage embryos were injected with 22.2 μM EnGen® Lba Cas12a (Cpf1) (New England Biolabs) and both gRNAs at a concentration of 13.8 mM each. All GFP+ larvae were raised to adulthood and germline-transmitting founders harboring the promoter deletion were identified by genotyping PCR with the following three primers: foxi3a-F: 5- CGATCAGAAAACGCCTGCAGACTGA-3’, foxi3a-R: 5’-GGGAGGTCTCACGAGTTTCATCAGATC-3’, foxi3a-R2: 5’-GCAACGAATGGAATCAGAATGTACAGTGC-3’. One F0 founder was outcrossed to wildtype fish to generate the F1 generation. F1 fish were raised to adulthood and heterozygous carriers of the deletion were identified by genotyping PCR. Multiple pairs of heterozygous F1 fish were incrossed and F2 larvae were analyzed for all experiments.

Peloggia J., Münch D., Meneses-Giles P., Romero-Carvajal A., Lush M.E., Lawson N.D., McClain M., Pan Y.A, & Piotrowski T. (2021). Adaptive cell invasion maintains lateral line organ homeostasis in response to environmental changes. Developmental cell, 56(9), 1296-1312.e7.

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CRISPR and ABE Knockout of CYP81A6 in Rice

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The gene-edited rice CYP81A6 knockouts by using CRISPR/Cas9 and adenine base editor (ABE) were made in our laboratory. The NuClean Plant Genomic DNA Kit was purchased from CWBIOTECH Co., Ltd. (Jiangsu, China). The PCR reagent was purchased from Takara Biology Co., Ltd. (Dalian, China). The DNA Constant Temperature Rapid Amplification Kit (Basic) was purchased from Weifang Anpu Future Biotechnology Co., Ltd. (Weifang, China). The HiScribe T7 High Yield RNA Synthesis Kit and EnGen Lba Cas12a (Cpf1) were purchased from New England Biolabs (Ipswich, MA UK). RNA Clean & Concentrator-25 was purchased from Zymo Research (California, USA). All of the primers were synthesized by Sangon Biotech (Shanghai, China). The polyethylene filter membrane (aperture, 10 μm), silica gel adsorption membrane (aperture, 1 μm), and screw column were purchased from Hangzhou Laifeng Biotechnology Co., Ltd. (Hangzhou, China).

Wang M., Liu X., Yang J., Wang Z., Wang H, & Wang X. (2022). CRISPR/Cas12a-based biosensing platform for the on-site detection of single-base mutants in gene-edited rice. Frontiers in Plant Science, 13, 944295.

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LAMP-based SARS-CoV-2 Detection Assay

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AuNP was obtained from Suzhou Tanfeng Graphene Technology Co., Ltd (Jiangsu, China). Nucleic acid sequences (Table S1) were ordered from Sangon Biotech (Shanghai, China). WarmStart® LAMP Kit (DNA & RNA), Bst 3.0 DNA Polymerase, EnGen® Lba Cas12a (Cpf1) and NEBuffer™ 2.1 were obtained from New England Biolabs (Beijing, China). Tris(2-carboxyethyl) phosphine (TCEP) was obtained from Aladdin (Shanghai, China). SYBR Green Ⅰ was purchased from Solarbio (Beijing, China). BM2000 + DNA Marker was obtained from Biomed (Beijing, China). DEPC water (DNase/RNase free) was obtained from Beyotime (Shanghai, China). TE Buffer and GeneJET RNA Purification Kit were obtained from Thermo Fisher Scientific (Shanghai, China). COVID-19 (Corona Virus Disease 2019) RNA reference material (high concentration) was obtained from the National Institute of Metrology of China.

Zhang Y., Chen M., Liu C., Chen J., Luo X., Xue Y., Liang Q., Zhou L., Tao Y., Li M., Wang D., Zhou J, & Wang J. (2021). Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP. Sensors and Actuators. B, Chemical, 345, 130411.

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CRISPR/Cas12a-FBDA for Rapid Visual Diagnostics

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The CRISPR/Cas12a-FBDA performed as described previously with minor modifications.[23 (link),24 (link)] The CRISPR/Cas12a reaction was composed of 30 nM of crRNA, 330 nM of EnGen Lba Cas12a (Cpf1) (New England Biolabs, USA), 600 nM of fluorescent probe (5′-FAM-TTATTATT-BHQ1-3′), 1X of NEBuffer 2.0 (New England Biolabs, USA), and 1 μL of RPA amplicons in a total reaction volume of 15 μL. The CRISPR/Cas12a reaction was incubated at 39°C for 20 min. The fluorescent signal was then observed by naked eye using a BluePAD Dual LED Blue/White Light Transilluminator (BIO-HELIX, Taiwan) at 470 nm wavelength. Each test was observed by three certified laboratory technicians who were instructed to identify the qualitative test outcome as positive or negative. The tests were considered positive if at least two of the three technicians read the results as positive. Both investigators and the technicians were unaware of the outcome.

Jirawannaporn S., Limothai U., Tachaboon S., Dinhuzen J., Kiatamornrak P., Chaisuriyong W., Bhumitrakul J., Mayuramart O., Payungporn S, & Srisawat N. (2022). Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32. PLoS Neglected Tropical Diseases, 16(1), e0010112.

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Isothermal Amplification with RPA-Cas12a

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Recombinase polymerase amplification Kits used for isothermal amplification were purchased from Lesunbio Co., Ltd. (Wuxi, China). Primers for RPA reaction, crRNA and double distilled water were ordered from Sangon Biotech Co., Ltd. (Shanghai, China). The ssDNA was synthesized by TianyiHuiyuan Biotechnology Co., Ltd. (Beijing, China). The 100 bp DNA ladder was obtained from TransGene Biotech Co., Ltd. (Beijing, China). EnGen^® Lba Cas12a (Cpf1) and NEBuffer™ r2.1 were purchased from New England BioLabs (Beijing, China). QIAamp DNA Mini Kits used for DNA extraction were obtained from Qiagen GmbH (Hilden, Germany).

Li F., Xiao J., Yang H., Yao Y., Li J., Zheng H., Guo Q., Wang X., Chen Y., Guo Y., Wang Y, & Shen C. (2022). Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection. Frontiers in Microbiology, 13, 858806.

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Integrated LAMP-CRISPR Detection System

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The LAMP system contained the same mixture as the real-time LAMP assay. The fluorescent dyes were added into the system and the LAMP reagents covered with 40 μL of mineral oil. The LAMP reaction was followed by the admixture of CRISPR reaction solution (preloaded inside the lid) to the LAMP amplification solution by centrifugation. The 20 μL CRISPR reaction solution contained 1 × NEBuffer 2.1, reaction buffer, 0.2 μM EnGen^® Lba Cas12a (Cpf1) (New England Biolabs), 0.6 μM gRNA, 2.5 μM CRISPR probes, and 1 U/μL RNA inhibitor (Thermo Fisher Scientific, Inc., Waltham, MA, United States). Sequence information of gRNA is shown in Table 1. The CRISPR reaction was conducted in a ProFlex PCR System (Applied Biosystems, Inc., Carlsbad, CA, United States) at 37°C for 5 min. After the reaction, the visualized results were observed using a simple fluorescence reader.

Chen Y., Shi Y., Zhu W., You J., Yang J., Xie Y., Zhao H., Li H., Fan S., Li L, & Liu C. (2021). Combining CRISPR-Cas12a-Based Technology and Metagenomics Next Generation Sequencing: A New Paradigm for Rapid and Full-Scale Detection of Microbes in Infectious Diabetic Foot Samples. Frontiers in Microbiology, 12, 742040.

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Enzymatic Modification of Biomolecules

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EnGen Lba Cas12a (Cpf1), 10X NEBuffer™ 2.1 buffer, Klenow fragment (3′ → 5′ exo-), 10X NEBuffer™ 2, deoxynucleotide (dNTP) solution mix, nicking endonuclease (Nb.BbvCI) were purchased from New England Biolabs Ltd. (Whitby, ON, Canada). Streptavidin from Streptomyces avidinii and biotin were purchased from Sigma (Oakville, ON, Canada). IL-6 protein and polyclonal anti-IL-6 antibodies were purchased from Thermo fisher Scientific (Mississauga, ON, Canada). Human serum, magnesium chloride hexahydrate (MgCl₂·6H₂O), and 100× Tris–EDTA (TE, pH 7.4) buffer were purchased from Sigma-Aldrich (Mississauga, ON, Canada). NANOpure H₂O (>18.0 MΩ), purified using an Ultrapure Mili-Q water system, was used for all experiments. All DNA samples and the guide RNAs were Integrated DNA Technologies (Coralville, IA) and purified using high-performance liquid chromatography.

Li Y., Mansour H., Watson C.J., Tang Y., MacNeil A.J, & Li F. (2021). Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay. Chemical Science, 12(6), 2133-2137.

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Isothermal CRISPR-Cas12a Nucleic Acid Extraction

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Genomic DNA kits for nucleic acid extraction and purification were purchased from Beijing TransGen Biotech. Co, Ltd (Beijing, China). The universal isothermal amplification kit was provided from HuiDeXin Biotech. Co., Ltd. (Tianjin, China). EnGen^® Lba Cas12a (Cpf1) and 10 × NE Buffer 2.1 were purchased from New England Biolabs (MA, USA). The real-time turbidimeter (LA-320C) was purchased from Eiken Chemical. Co, Ltd (Japan). The ABI7500 real-time fluorescent quantitative PCR systems was purchased from Applied Biosystems (USA). The BluSight Pro (GD50502) was obtained from Manod Biotech. Co, Ltd (Suzhou, China).

Jia N., Wang C., Liu X., Huang X., Xiao F., Fu J., Sun C., Xu Z., Wang G., Zhou J, & Wang Y. (2023). A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application. Frontiers in Cellular and Infection Microbiology, 13, 1192134.

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CRISPR-based Detection of Viral RNA

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35 μL of mineral oil was covered on the RT-LAMP reaction solution (The reaction system was the same as real time RT-PCR except that the fluorescent dyes were replaced with water). After RT-LAMP reaction, 20 μL of CRISPR reaction solution (pre-added inside the lid) was mixed with the RT-LAMP amplification solution by hand shaking. 20 μL CRIPSR reaction solution contained 1 × NEBuffer 2.1 Reaction Buffer, 0.2 μM EnGen® Lba Cas12a (Cpf1) (New England Biolabs Inc., Ipswich, MA, USA), 0.6 μM gRNA, 2.5 μM CRISPR probes, 1 U/μL RNA inhibitor (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The tube was put in ProFlex PCR System (Applied Biosystems, Inc., Carlsbad, CA, USA) at 37 °C for 5 min. After the reaction, 3D printing instrument and smart phone were used to observe the fluorescence.

Chen Y., Shi Y., Chen Y., Yang Z., Wu H., Zhou Z., Li J., Ping J., He L., Shen H., Chen Z., Wu J., Yu Y., Zhang Y, & Chen H. (2020). Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection. Biosensors & Bioelectronics, 169, 112642.

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Urease-Based Biosensing Assay Protocol

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urease
powder from Canavalia ensiformis (jack
bean), phenol red, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(Sulfo-SMCC), urea, sodium chloride, polysorbate 20 (Tween 20), and
ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma-Aldrich
(St. Louis, MO, USA). Synthetic oligonucleotides used in this study,
their sequences, and functions are listed in Table S1. Modified DNA oligonucleotides were purchased from Bioneer
(Daejeon, Republic of Korea) and Integrated DNA Technologies (Coralville,
IA, USA). M-270 streptavidin magnetic beads were obtained from Thermo
Fisher Scientific Inc. (Waltham, MA, USA). Dulbecco’s phosphate-buffered
saline (D-PBS) (1×) was purchased from Welgene (Gyeongsan, Republic
of Korea). EnGen Lba Cas12a (Cpf1) was purchased from New England
BioLabs (Ipswich, MA, USA). Forty percent acrylamide/bis solution
(19:1) was purchased from Bio-Rad (Hercules, CA, USA). Tris–HCl
(1 M; pH 7.2) was purchased from Biosesang (Seongnam, Republic of
Korea). A Synergy H1 hybrid multimode microplate reader and a Cytation
hybrid multimode reader (Winooski, VT, USA) were used to measure fluorescence.

Ki J., Na H.K., Yoon S.W., Le V.P., Lee T.G, & Lim E.K. (2022). CRISPR/Cas-Assisted Colorimetric Biosensor for Point-of-Use Testing for African Swine Fever Virus. ACS Sensors, 7(12), 3940-3946.

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