Cas12a Nuclease (CRISPR-associated endonuclease Cas12a)

I. Introduction

Cas12a (also known as Cpf1) is a Class 2, Type V CRISPR effector protein mediated by crRNA. It is a site-specific DNA endonuclease guided by a single crRNA.

Compared with the widely used Cas9 system, Cas12a has the following unique properties:

(1) Single guide RNA: No tracrRNA required; guided solely by a single crRNA.

(2) Distinct PAM recognition: Recognizes T-rich PAM sequences such as 5'-TTN-3' or 5'-TTTV-3' (V = A/C/G), in contrast to the G-rich PAM required by Cas9.

(3) Low off-target effect: Cas12a achieves comparable genome-editing efficiency to Cas9 with lower off-target rates.

(4) Generates sticky ends: Cleavage of target double-stranded DNA produces sticky ends, facilitating precise gene editing.

(5) Trans-cleavage activity: Upon binding to target DNA, its non-specific ssDNA trans-cleavage (collateral cleavage) activity is activated, enabling cleavage of single-stranded DNA reporter probes in the system.

II. Product Information

1. Enzyme Activity: Benchmark against similar products

2. Specifications

III. Storage Conditions & Shelf Life

(1) Storage temperature: Store at -20℃; -80℃ recommended for long-term storage.

(2) Shelf life: Stable for 1 year at -20℃.

(3) Notes: Aliquot according to usage frequency to avoid repeated freeze-thaw cycles.

IV. Protocol

Place Cas12a, gRNA and substrate DNA on ice. Dilute gRNA to 300 nM and substrate DNA to 30 nM with nuclease-free water.

2. Reaction system (30 μL example):

Component	Volume	Working Concentration
Nuclease-free Water	20 μL
10X Reaction Buffer	3 μL
gRNA (300 nM)	3 μL	0.9 pmol
Cas12a (1 μM)	1 μL	1 pmol
Total Volume	27 μL

3. Gently mix by pipetting or brief low-speed vortex, spin down briefly at room temperature to collect liquid at tube bottom. Pre-incubate at 25℃ for 10 min.

4. Add 3 μL of 30 nM target substrate DNA (final volume 30 μL), mix gently by pipetting or low-speed vortex, spin down, then incubate at 37℃ for 10 min.

5. Add 1 μL Proteinase K to each sample, mix gently and incubate at room temperature for 10 min.

6. Add 6 μL 6X DNA Loading Buffer, then perform electrophoresis analysis using 1% agarose gel.

Precautions

1. Nuclease-free conditions must be maintained during handling. Take special care to avoid contamination. All operations should follow RNase-free requirements.

2. This product is for scientific research use by qualified professionals only. Not for clinical diagnosis or therapy, food or pharmaceutical applications. Do not store in residential premises.

3. Wear lab coat and disposable gloves for personal safety during operation.

Product	Cas12a Nuclease
Source
Molecular Weight	147.9 kDa
Appearance	Colorless and transparent liquid
Concentration	44.43 nmol/mL
Pack Size	44.43 nmol/vial
Purity
Storage Buffer	40 mM Tris, 300 mM NaCl, 5% Glycerol, pH 7.5;
Inactivation	Heat at 65℃ for 10 min for inactivation.