EB0502 LbCas12a

LbCas12a

Cat#EB0502

Description

So far, six types and more than 20 subtypes of CRISPR-Cas systems have been identified.CRISPR-Cas12a is the second class (Type V) of CRISPR-Cas systems used to edit mammalian genomes.Cas12a has unique capabilities that complement the CRISPR-Cas9 system. The Cas12a (Cpf1) protein has a RuvC endonuclease domain similar to Cas9.

In addition, Cpf1 does not have an HNH endonuclease domain, and the N-terminal of Cas12a does not have the alpha-helical recognition lobe of Cas9. The double-strand DNA break after Cas12a performs the cut introduces 4 or 5 nucleotide protruding sticky ends.

The binding of the LbCas12a-crRNA complex to complementary single-stranded ordoublestranded DNA releases powerful non-specific ssDNA trans-cleavage activity, which makes Cas12a a novel tool for genetic detection and imaging.

Product Features

LbCas12a (also known as LbCpf1) is a DNA endonuclease guided by crRNA alone, derived from Lachnospiraceae bacterium strain and obtained by recombinant expression of Escherichia coli.

LbCas12a, in contrast to Cas9 which requires G-rich PAM, cleaves target DNA by recognising T-rich (5'-TTTN-3') PAM motifs.

LbCas12a is smaller than Cas9, and LbCas12a does not require tracrRNA, but only requires crRNA molecules with smaller crRNA (close to half of the total sgRNA of Cas9), which is very conducive to genome editing.

Product Composition

Quality Control

Protein Purity

The purity of LbCas12a was determined by molecular exclusion high performance liquid phase.

Endonuclease Activity

The 20 μL reaction system consisted of 10 pmol LbCas12a and 0.6 μg λ-Hind III were incubated at 37℃ for 3 hours, and the electrophoretic bands of DNA did not change.

Exonuclease activity

The 20 μL reaction system consisted of 10 pmol LbCas12a and 0.6 μg λDNA were incubated at 37℃ for 4h, and the electrophoretic bands of DNA did not change.

RNase residues

The 20 μL reaction system consisted of 10 pmol of LbCas12a and 2 μg RNA were incubated at 37℃ for 1h, and the electrophoretic bands of RNA did not change.

E. coli DNA residues

In a 20μL reaction system, E. coli 16 s rDNA gene was amplified using LbCas12a as a template, and no amplification was achieved within 30 cycles.

Usages

Non-viral vector CRISPR gene editing;

Specific site cutting of target DNA in vitro;

Novel genetic testing and imaging.

Storage Conditions

< 0℃ transport; Storage at -20℃, long-term storage at -80℃, to avoid repeated freezing and thawing.

It is valid for 12 months at -20℃.

Suggested Usage Methods

1. The crRNA sequence design of LbCas12a is referenced below:

5′–AAUUUCUACUAAGUGUAGAUNNNNNNNNNNNNNNNNNNNN–3′。

Among other things, the crRNA of LbCas12a contains direct repeat sequences (DR region, aka Scaffold region, underlined), and guide sequences with a length of 20 nt-35 nt that are complementarily paired with specific target sequences (Spacer region, indicated in blue font).

2. Inverse shear experiments, suggested reaction system:

*Target DNA may be ssDNA or dsDNA with PAM sequence.

3. Real-time fluorescence quantitative PCR instrument to detect fluorescence signal, 37 ℃ reaction, every 30 sec to collect fluorescence signal.

Examples of applications