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32117 LwaCas13a (C2c2) Nuclease

  • 32117 LwaCas13a (C2c2) Nuclease.png
  • 32117.jpg

32117 LwaCas13a (C2c2) Nuclease

  • Specifications:
    • 100 pmoL
    • 1,000 pmoL

Article number:

32117


Price:Contact our distributors


隐藏域元素占位

Product overview

Description

LwaCas13a (previously known as LwaC2c2) is a crRNA-mediated RNA endonuclease derived from Leptotrichia wadeim. It can specifically recognize and cleave the RNA target with the PFS sequence. In addition, Cas13a also has the trans-cleavage activity, that is, when LwaCas13a binds crRNA and RNA target to form a ternary complex, it releases the trans-cleavage activity of Cas13a against non-target ssRNA sequences. This activity has also been applied in the development of rapid nucleic acids detection systems. For example, Prof. Feng Zhang's team from the Broad Institute employed the Cas13 trans-cleavage activities and successfully developed the "SHERLOCK" platform (for details, please refer to PMID: 28408723).

 

 

Product Components

Components

32117-01 (100 pmol)

32117-03 (1,000 pmol)

LwaCas13a Nuclease (10 μM) a

100 pmol

1,000 pmol

10 × HOLMES Buffer 2

1 mL

1 mL

a.The concentration of LwaCas13a Nuclease is 10 μM (10 pmol/μL);

 

 

Storage Conditions

Store at -20℃; transport at ≤0℃.

 

 

Experimental Results

The tested trans-cleavage system in this experiment contained LwaCas13a, crRNA, ssRNA reporters, and RNA target of different concentrations.  The reporters were labeled with a fluorophore (FAM) at the 5'-end and a quencher (BHQ1) at the 3'-end. The results showed LwaCas13a released the trans-cleavage activity against ssRNA and cleaved the ssRNA reporters to emit fluorescent signals at the presence of RNA target. The higher concentrations of the RNA target, the faster fluorescence signals produced, and the shorter time to reach the plateau.

   

Fig 1. Results of Cas13 trans-cleavage at different concentrations of ssRNA target.

 

 

Experiment Procedure

1.Trans-cleavage experiment

Components

Volume 

Final concentration 

10 × HOLMES Buffer 2

2 μL 1 ×

10 μM LwaCas13a Nuclease

0.05~0.5 μL 25~250 nM

10 μM crRNA

0.05~0.5 μL 25~250 nM

10 μM Target RNA

0.05~0.5 μL 25~250 nM

10 μM ssRNA Reporter (FAM-BHQ1) 

0.05~0.5 μL 25~250 nM

Nuclease-free water

Up to 20 μL  

◆ The amount of each component in the trans-cleavage system can be adjusted according to different experimental purposes. When the amount is small, each component can be diluted first and then added to the system. LwaCas13a Nuclease can be diluted with 1×HOLMES Buffer 2, and it must be used immediately after dilution (If you want to store the diluted Cas13 enzyme for a long term, please use Cas13 Dilution Buffer, #32013). crRNA, Target RNA and ssRNA Reporter can be diluted with Nuclease-free Water, but for extremely low concentrations of Target RNA (such as LOD experiments), it is recommended to dilute with 0.1 % Tween 20 and use low adsorption centrifuge tubes and tips etc.

◆ For the amount of probe used in the trans-cleavage system and the expected reaction time to reach the plateau, please refer to the specific value of transU of each batch of Cas enzyme. For details, please refer to the COA test report provided by Tolo Biotech.

◆ The real-time fluorescent quantitative PCR instrument detects the fluorescent signal from reaction at 37°C, and collects the fluorescent signal every 30 seconds.

FAQ

Q1: What are the common Cas13 enzymes, and how do they differ from the Cas12 and Cas9 families?

A1: Common Cas13 enzymes include Cas13a, Cas13b, Cas13c etc. Cas13a/Cas13c have spacer regions at the 3' end of crRNA, such as LwaCas13a, while Cas13b has a spacer region at the 5' end of crRNA, such as PsmCas13b. In general, Cas13 proteins only require crRNA for guidance and recognize and cleave target ssRNA. Furthermore, ssRNA targets can activate Cas13's trans-cleavage activity, allowing for non-specific cleavage of ssRNA probes in the system. A detailed comparison is provided in the table below:

 

Cas9 

 Cas12

Cas13

Classification

Class II Type II

Class II Type V

Class II Type VI

Nuclease Domains

RuvC and HNH

RuvC only

HEPN × 2

Guide RNA

crRNA and tracrRNA

crRNA only or
crRNA and tracrRNA

crRNA only

PAM Sequence

5´-NGG-3´

T-Rich

/

Cis-Cleavage Target

dsDNA with PAM

dsDNA with PAM or ssDNA

ssRNA

Trans-Cleavage Target

/

ssDNA

ssRNA

 

Q2: How to design the crRNA for LwaCas13a?

A2: The crRNA for LwaCas13a can be designed based on the following sequence:
5′–GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACNNNNNNNNNNNNNNNNNNNN–3′, (Black represents the direct repeat region, and the blue underlined portion indicates the spacer region that complements the specific target sequence. It is recommended to have a spacer region of 28 nt in length).

 

 

Product Instruction

#32117 LwaCas13a Nuclease

pdf

574.8KB

References

COA Query

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