3 μL of 100 μM LbaCas12a endonuclease (NEB, M0653T), 5 μL of 20 μM gRNA1 (matching the target RNA sequence) and 10 μL of 20 μM gRNA2 (matching the Cir-mediator) was mixed at 3.6 mL 1× NEBuffer 2.1 and followed by adding 120 μL of 100 μM Texas Red quenched reporter and 36 μL of 1 M DTT. Afterwards, the prepared Cir-mediator solution was mixed to a final concentration of 50 nM before the use to form the final AUTOCAR reaction mixture for RNA. Then, 10 μL different concentration of target RNA (RNA or SARS-CoV-2 genome RNA) was mixed with 90 μL of the prepared final c-Car reaction mixture. The reaction was set at room temperature for 1–1.5 h, and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).
Lbacas12a endonuclease
Manufactured by New England Biolabs
LbaCas12a endonuclease is a CRISPR-associated enzyme derived from Lachnospiraceae bacterium. It functions as a programmable RNA-guided DNA endonuclease, capable of cleaving target DNA sequences in a sequence-specific manner.
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Lab products found in correlation
7 protocols using lbacas12a endonuclease
1
AUTOCAR Assay for RNA Detection
2
CRISPR/Cas12a Trans-Cleavage Activation Assay
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1 μL of 100 μM LbaCas12a endonuclease (NEB, M0653T) and 5 μL of 20 μM gRNA was mixed with 3.6 mL 1× NEBuffer 2.1, followed by addition of 6 μL of 100 μM Texas Red quenched reporter. The prepared standard CRISPR/Cas12a reaction mixture was stored at 4 °C for future use. For each CRISPR/Cas12a trans-cleavage activation reaction, 10 μL 10 nM of nucleic acid (L-ssDNA, L-dsDNA, Cir-ssDNA or Cir-mediator) with complementary sequence to gRNA was added into 90 μL of the prepared reaction buffer. The reaction was carried out at room temperature, and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).
3
Cas12a Triggered Trans-Cleavage Assay
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1 μL of 100 μM LbaCas12a endonuclease (NEB, M0653T) and 5 μL of 20 μM gRNA was mixed with 3.6 mL 1× NEBuffer 2.1, and followed by the adding of 100 nM of prepared DNA oligo (linear dsDNA, linear ssDNA, Cir-ssDNA or Cir-mediator). Then, 1 μM of trigger ssDNA oligo was added to activate Cas12a trans-cleavage for 60 min at room temperature. Afterwards, 10 μL of the trans-cleavage product was transferred into 90 μL of the standard CRISPR/Cas12a reaction mixture with gRNA matching the Cir-mediator, and set at room temperature for 60 min. The fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).
4
Sensitive DNA Detection with AutoCAR-1
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3 μL of 100 μM LbaCas12a endonuclease (NEB, M0653T), 5 μL of 20 μM gRNA1 (matching the target DNA sequence) and 10 μL of 20 μM gRNA2 (matching the Cir-mediators) was mixed at 3.6 mL 1× NEBuffer 2.1 and followed by adding 12 μL of 100 μM Texas Red quenched reporter. Afterwards, the prepared Cir-mediator solution was mixed to a final concentration of 50 nM to form the final AutoCAR-1 reaction mixture for DNA detection. Then, 10 μL of different concentrations of target DNA (ssDNA, dsDNA or H. pylori genome DNA) were mixed with 90 μL of the prepared final AutoCAR-1 reaction mixture. The reaction was set at room temperature for 1 h, and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA).
5
AUTOCAR: Rapid Fluorescent Assay
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3 μL of 100 μM LbaCas12a endonuclease (NEB, M0653T), 5 μL of 20 μM gRNA1 (for trigger DNA/RNA) and 10 μL of 20 μM gRNA2 (for Cir-mediator) was mixed with 3.6 mL of 1× NEBuffer 2.1 and followed by the addition of 12 μL of 100 μM Texas Red quenched reporter. Afterwards, the prepared Cir-mediator solution was mixed to a final concentration of 50 nM before use to form the final AUTOCAR reaction mixture. Then, 10 μL of different concentrations of trigger DNA (ssDNA or dsDNA) with a complementary sequence to gRNA was mixed with 90 μL of the prepared final AUTOCAR reaction mixture to initiate the reaction. The reaction was set at room temperature and the fluorescence intensity at Ex/Em of 570/615 nm was determined by using a plate reader (iD5 Spectramax, Molecular Devices, USA). Alternatively, Cir-mediator can be replaced by 50 nM of the same volume of 1× NEBuffer 2.1 for a comparison with a standard Cas12a reaction as per Method 2 above.
6
CRISPR/Cas12a Cleavage Kinetics
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1 μL of 100 μM LbaCas12a endonuclease (NEB, M0653T) and 5 μL of 20 μM gRNA was mixed with 3.6 mL 1× NEBuffer 2.1, and followed with the adding of 1 μM non-trigger ssDNA or dsDNA oligo. Then, 10 μL of 1 μM trigger ssDNA was mixed with 90 μL of the prepared standard CRISPR/Cas12a reaction mixture. The reaction was set at room temperature for 0–60 mins, and each of 10 μL cleavage product from 0, 10, 20, 30, 40, 50, 60 min was mixed with 2 μL of 6X DNA gel loading dye, and then loaded onto 4% agarose gel for electrophoresis at a constant voltage of 80 V for 60 min. Gel images were visualized by using Gel Doc + XR image system (Bio-Rad Laboratories Inc., USA).
7
CRISPR/Cas12a Nuclease Activation with Antibody-ssDNA Conjugate
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Preparation of the standard CRISPR/Cas12a reaction mixture: 10 μL 10 μM of LbaCas12a endonuclease (NEB, M0653T) was mixed into 3.6 mL milliQ water diluted 1X NEB 2.1 buffer. Then, 5 μL of 20 μM synthesized gRNA and 6 μL of 100 μM of ssDNA linked fluorescentquenched reporter (Table S1) were gently mixed to form the final reaction mixture. This mixture was stored at 4 • C for further use.
To verify the capability of the prepared Abs-ssDNA for triggering nuclease activity of CRISPR/Cas12a, 5 μL of the prepared Abs-ssDNA conjugate, or 5 μL of free triggering ssDNA have been mixed with μL of the standard CRISPR/Cas12a reaction mixture. Then, after 1-h incubation at room temperature, the fluorescent signal intensity has been measured by an iD3 plate reader at Ex = 570 nm, Em = 615 nm. Also, to verify the universality of making the Abs-ssDNA conjugate, different additional antibodies (anti-IL-1β, anti-IL-6, anti-EGFR) have separately been used to form the Abs-ssDNA, followed by the same Y. Li et al.
previously described protocol. Then, 5 μL of each prepared Abs-ssDNA has been mixed with 100 μL of standard CRISPR/Cas12a reaction mixture. After 1-h incubation at room temperature, the fluorescent signal has been tested by an iD3 plate reader at Ex = 570 nm, Em = 615 nm.
To verify the capability of the prepared Abs-ssDNA for triggering nuclease activity of CRISPR/Cas12a, 5 μL of the prepared Abs-ssDNA conjugate, or 5 μL of free triggering ssDNA have been mixed with μL of the standard CRISPR/Cas12a reaction mixture. Then, after 1-h incubation at room temperature, the fluorescent signal intensity has been measured by an iD3 plate reader at Ex = 570 nm, Em = 615 nm. Also, to verify the universality of making the Abs-ssDNA conjugate, different additional antibodies (anti-IL-1β, anti-IL-6, anti-EGFR) have separately been used to form the Abs-ssDNA, followed by the same Y. Li et al.
previously described protocol. Then, 5 μL of each prepared Abs-ssDNA has been mixed with 100 μL of standard CRISPR/Cas12a reaction mixture. After 1-h incubation at room temperature, the fluorescent signal has been tested by an iD3 plate reader at Ex = 570 nm, Em = 615 nm.
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