Application

Functional Genomics/Target Validation/Genome Editing/Molecular Biology/Molecular Cloning/Synthetic Biology

Components

Each kit consists of: one vial of FnCas9 recombinant protein one vial containing 1 mL of 1× Dilution buffer one vial containing 1 mL of Nuclease free water with glycerol one vial containing 1 mL of 10× Digestion Buffer

Features and Benefits

Suitable for plasmid or large genomic DNA fragment cloning Results in 4-bp 5’; overhangs. Cuts purified plasmid DNA more efficiently than SpCas9

General description

The purified FnCas9 protein (~190 kDa) is derived from the type II CRISPR/Cas system from the bacterium Francisella novicida and has been engineered to function using three molecular components: the FnCas9 protein, a crRNA of user-defined sequence, and a tracrRNA of constant sequence. The FnCas9 endonuclease can be programmed by changing the crRNA sequence to create a site-specific double-strand break (DSB) in plasmids or purified genomic DNA.

Legal Information

MISSION is a registered trademark of Sigma-Aldrich Co. LLC

CRISPR Use License Agreement

Other Notes

Click here to order gRNAEnter the 20bp genomic DNA target sequence (5′ to 3′) plus the adjacent 3′ PAM site. Under special instructions, indicate 'FnCas9 crRNA'Click here to order FnCas9 tracrRNACheck out our other MISSION^® Cas9 Proteins at SigmaAldrich.com/CRISPRproteins

Packaging

pkg of 50 µg (≥ 300 pmol)pkg of 250 µg (≥ 1500 pmol)

Principle

Conventional restriction enzymes such as EcoRI and HindIII can only cut specific short (6 bp) DNA sequences and are therefore limited to cloning small DNA fragments (typically ~4 kb). CRISPR-based endonculeases can cut user defined sequences of a longer length (~20 bp) making them more flexible and applicable to larger DNA fragments such as multigene bacterial operons or mammalian genomic DNA with native promoter, exon, and intron components.

Reconstitution

Lyophilized FnCas9 protein should be resuspended in the Reconstitution solution provided to desired concentration. Gently tap tube to completely dissolve lyophilized powder, incubate for 10 minutes on ice, and spin tube to bring material to bottom of tube.