Cas12 Proteins

CRISPR Cas12 Proteins for High Fidelity DNA Cleavage

Cas12 Proteins (CRISPR Cas Type V Nucleases) are a highly attractive alternative to Cas9 Nucleases as they reveal higher specificity via trans-cleaving non specific ssDNA, which minimizes off-target effects, and enables molecular precision diagnostics by employing ssDNA sensors.

Cas12 nucleases produce staggered double strand breaks in contrast to Cas9 which creates blunt ends, use a different PAM (Protospace Adjacent Motif), and need to be complexed with a variety of sgRNA designs, depending on the Cas12 variant.

Nucleus Biotech is pleased to offer AapCas12b-NLS from  Alicyclobacillus acidiphilus for High Fidelity CRISPR Genome Editing, and AapCas12b as well as AsCas12a (from Acidaminococcus spec.) , LbCas12a (from Lachnospiraceae spec.), and FnCas12a (from Francisella novicida) without NLS for CRISPR-based high accuracy gene detection technologies. FnCas12a has also been shown to be very suitable for targeted genome editing in bacteria.

Recombinant Cas12b-NLS Protein for High-Fidelity CRISPR Genome Editing Applications

• Recombinant AapCas12b for forming RNP complexes with guide-RNA

• Highly purified and extremely active (staggered double strand DNA cleavage)

• Trans ssDNA cleavage activity leads to highly increased specificity

• NLS for delivery into the nucleus and high efficiency genome editing

Cas12b Nucleases belong to type V CRISPR effectors and are a highly attractive alternative to Cas9 Nucleases for engineering mammalian genomes as they reveal higher specificity via trans-cleaving non specific ssDNA, which minimizes off-target effects. They are also smaller in size, which makes them more suitable for potential gene therapy approaches, and they tolerate a wide temperature range, which is ideal for biomedical gene detection techniques.

Nucleus Biotech is pleased to offer Trialtus Biosciences´ recombinant AapCas12b originating from Alicyclobacillus acidiphilus (see Teng et al., Nature, Cell Discovery volume 4, Article number: 63 (2018)  for single and multiplex genome editing, gene activation, and generation of gene mutants with CRISPR technology and for CRISPR-mediated in vitro applications.

TriAltus’ highly pure recombinant AapCas12b protein combines with suitable guide RNA (gRNA) to form a stable ribonucleoprotein (RNP) complex that enables the inactive nuclease to be guided to the specific double stranded DNA (dsDNA) site. Once guided to the specific cleavage site, AapCas12b becomes activated to specifically and precisely cleave the dsDNA and to indiscriminately trans-cleave single stranded DNA (ssDNA) nearby.

Trialtus Biosciences´ protein is purified tag-free by their proprietary CL7 tag CLiM technology, which ensures extremely high purity and activity:

AapCas12b protein is expressed in E. coli with two C-terminal nuclear localization signals (NLS) and the CL7 tag, which is removed with PreScission protease during the purification process.

CLīM-purified AapCas12b’s trans-cleavage nuclease activity compared to commercially obtained (“C.O.”) AapCas12b. Upon hybridization of the Cas12b/sgRNA RNP to the target, dsDNA activator, Cas12b becomes active to cleave the activator as well as to trans-cleave nearby nonspecific ssDNA. Activated Cas12b cleaves dual-labeled fluorophore and quencher (FQ)-ssDNA Reporter, which releases the fluorophore to emit a fluorescent signal. Raw RFU values from each reaction were corrected by subtracting the background fluorescence value of the appropriate “no activator” control. The average of the corrected RFU values and SEM are reported. Each condition was performed in technical triplicates.
AapCas12b obtained from a commercial source or CLīM-purified AapCas12b was combined with annealed sgRNA for 10 minutes at room temperature to create functional AapCas12b RNP complexes. Reaction buffer was used in the place of protein for the “No Cas12b” control. A mixture of target dsDNA activator and a nonspecific FQ-ssDNA reporter was added to the designated RNP reactions. The reaction proceeded for 30 minutes at 37°C, equilibrated to room temperature for 10 minutes, and the fluorescence was measured. The reaction systems comprised the following: 0 or 40nM AapCas12b, 44nM sgRNA, 0 or 1 nM Activator, 100 nM FQ-Reporter and 1x NEBuffer r2.1 (New England Biolabs).

Recombinant Cas12 Proteins for Precision Gene Detection Technologies

Recently developed CRISPR-Dx molecular diagnostics methods based on Cas12 and Cas13 possess great potential for rapid and convenient nucleic acid detection.

Guided by specially designed sgRNAs, Cas12 nucleases bind target DNA sequences extremely specifically and trans-cleave ssDNA reporters for generation of detectable signals. The highly precise detection system can be combined with pre-amplification procedures including isothermal amplifications such as LAMP, to achieve one-pot reaction systems, suitable for field-testing, and offering many advantages over qPCR-based detection.