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32116 AacCas12b (C2c1) Nuclease

  • 32116 AacCas12b (C2c1) Nuclease.png
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32116 AacCas12b (C2c1) Nuclease

  • Specifications:
    • 100 pmoL
    • 1,000 pmoL

Article number:

32116


Price:Contact our distributors


隐藏域元素占位

Product overview

Description

AacCas12b (previously known as AacC2c1) is a DNA endonuclease mediated by tracerRNA and crRNA (or sgRNA), which can specifically cleave the dsDNA target in a PAM (TTN)-dependent manner, generating DSBs, and cleave ssDNA target in a PAM-independent manner. In addition, both dsDNA and ssDNA targets can trigger the trans-cleavage activity of AacCas12b, that is, when Cas12b binds sgRNA and target DNA to form a ternary complex, the trans-cleavage activity of Cas12b will be released to non-specifically cleave ssDNA sequences in the reaction system. AacCas12b is derived from Alicyclobacillus acidoterrestris, and the optimal reaction temperature is 48°C for AacCas12b. In addition, AacCas12b can be used not only for specific cleavage of dsDNA in vitro, but also for rapid detection of target nucleic acid. For details, please refer to the HOLMESv2 nucleic acid detection technology developed by Tolo Biotech (for details, please refer to PMID: 31532637).

 

 

Product Components

Components

32116-01 (100 pmol)

32116-03 (1,000 pmol)

AacCas12b Nuclease (10 μM) a

100 pmol

1,000 pmol

10 × HOLMES Buffer 1

1 mL

1 mL

a.The concentration of AacCas12b Nuclease is 10 μM (10 pmol/μL);

 

 

Storage Conditions

Store at -20℃; transport at ≤0℃.

 

 

Experimental Results

Fig1. Cis-cleavage.

 

Fig2. Trans-cleavage.

 

Experiment Procedure

1.Cis-cleavage

Components

Volume 

Final concentration 

10 × HOLMES Buffer 1

2 μL 1 ×

10 μM AacCas12b Nuclease

0.5 μL 250 nM

10 μM sgRNA

0.5 μL 250 nM

1 μM Target DNA

0.5 μL 25 nM

Nuclease-free water

Up to 20 μL  

◆ Target DNA can be ssDNA or dsDNA with PAM sequence in the cis-cleavage system.

◆ If nucleic acid electrophoresis is used to analyze cis-cleavage products, it is recommended to use dsDNA targets because ssDNA targets will be further cleaved by trans-cleavage-activated Cas12b. For a 20 μL cis-cleavage reaction system, the recommended amount of target DNA is 100 ng to 500 ng, and the length of the target DNA fragment is in the range of 300 bp to 3 kb. The amount of Cas enzyme and sgRNA required for different lengths of target DNA needs to be calculated according to the molar amount of target DNA. It is recommended to keep the molar ratio of Cas enzyme:sgRNA:target DNA at 10:10:1 to ensure that the target DNA is cleaved completely.

◆ Incubate at 48°C for 30 min~1 h, inactivate at 85°C for 5 min, then analyze the cis-cleaved products by nucleic acid electrophoresis.

 

2. Trans-cleavage experiment

Components

Volume 

Final concentration 

10 × HOLMES Buffer 1

2 μL 1 ×

10 μM AacCas12b Nuclease

0.05~0.5 μL 25~250 nM

10 μM sgRNA

0.05~0.5 μL 25~250 nM

1 μM Target DNA

0.5~5 μL 25~250 nM

10 μM ssDNA Reporter

0.05~0.5 μL 25~250 nM

Nuclease-free water

Up to 20 μL  

◆ Target DNA can be ssDNA or dsDNA with PAM sequence in the trans-cleavage system.

◆ 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. AacCas12b Nuclease can be diluted with 1×HOLMES Buffer 1, and it must be used immediately after dilution (If you want to store the diluted Cas12 enzyme for a long term, please use Cas12 Dilution Buffer, #32012). sgRNA, Target DNA and ssDNA Reporter can be diluted with Nuclease-free Water, but for extremely low concentrations of Target DNA (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 48°C, and collects the fluorescent signal every 30 seconds.

FAQ

Q1: What are the common Cas12b enzymes, and how do they differ from the Cas12a family?

A1: The Cas12b family of enzymes is typically more heat-resistant than the Cas12a family. Common Cas12b enzymes, such as AacCas12b, can function within a temperature range of 37°C to 55°C, with an optimum temperature of 48°C. AapCas12b, on the other hand, is even more heat-resistant and can function in a temperature range of 37°C to 65°C, making it suitable for use with LAMP (60°C to 65°C).

 

Q2: How to design the sgRNA for AacCas12b?

A2: The crRNA for AacCas12b consists of a 20-nt guide sequence (spacer region) and a 14-nt repeat sequence. The tracrRNA sequence includes a 16-nt anti-repeat region and three stem-loops, with a total length of approximately 91 nt. A fusion sgRNA, which retains the repeat region of crRNA and the anti-repeat region of tracrRNA, has a length of about 110 nt and can maintain similar efficiency to crRNA:tracrRNA co-guided systems. The sgRNA for AacCas12b can be designed based on the following sequence:
5′– GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACNNNNNNNNNNNNNNNNNNNN–3′, 

(The underlined portion represents the spacer region that complements the specific target sequence).

 

Q3: How should the ratio of Cas12b enzyme: sgRNA: target DNA be adjusted in the cis-cleavage system?

A3: To ensure complete cleavage of the target dsDNA, it is conventionally recommended to maintain a Cas12b enzyme: sgRNA: target DNA ratio of 10:10:1. If sgRNA efficiency is low, it is suggested to try a Cas12b enzyme: sgRNA ratio of 1:1.25~2, as excessive sgRNA can also negatively affect Cas12b cleavage. Additionally, if the sgRNA is prepared by transcription, it is important to ensure the purity of the transcription product.

 

Q4: Where are the cleavage sites for AacCas12b on dsDNA targets, and what determines them?

A4: The Cas12b family recognizes PAM sequences rich in T (5′-TTN-3′), and the cleavage sites on dsDNA targets are determined by the presence of a PAM sequence. In the case of AacCas12b, its cleavage sites on the non-target strand (NTS) are around position 17 from the PAM, and on the target strand (TS), they are around position 24 from the PAM. Cleavage occurs in an alternating fashion, resulting in a 5' overhang of 7 nt.

 

Q5: Can both dsDNA and ssDNA targets activate AacCas12b's trans-cleavage activity, and which is more efficient?

A5: Both dsDNA and ssDNA targets can activate AacCas12b's trans-cleavage activity, similar to Cas12a. However, ssDNA targets typically stimulate the trans-activity of Cas12b more efficiently, while dsDNA targets usually activate the trans-activity of Cas12a more effectively.

 

Q6: Does AacCas12b have sequence preferences for its trans-cleavage activity?

A6: There are reports that Cas12b enzymes cleave PolyA, PolyT, and PolyC sequences more efficiently than PolyG sequences. This suggests that Cas12b proteins have sequence-dependent preferences for trans-cleaving ssDNA substrates. The specific preferences would need to be verified through experiments.

 

Product Instruction

#32116 AacCas12b Nuclease

pdf

645.4KB

References

COA Query

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