FnCas9 plasmid

This product is an expression plasmid that utilizes the CMV promoter for strong transient expression of Francisella novicida Cas9 (CMV-FnCas9). The FnCas9 expression plasmid is one part of a two part CRISPR system with individual FnCas9 and gRNA expression vectors.

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Functional Genomics/Target Validation

• Proxy CRISPR
• Creation of gene knockouts in multiple cell lines

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II-B CRISPR/Cas system from the bacterium Francisella novicida has been engineered to function in eukaryotic systems using two molecular components: a single FnCas9 nuclease and a non-coding guide RNA (gRNA). The FnCas9 endonuclease is human codon optimized and programmed with a gRNA, directing a DNA double-strand break (DSB) at a desired sequence of DNA. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB. FnCas9 is similar to common nucleases in many ways but also possess other distinct qualities. It has been determined to possess a higher intrinsic specificity through a reduced tolerance to gRNA:DNA mismatches as well as inducing a staggered cleavage pattern leaving 4bp 5′ overhangs by cleaving the non-target strand at 7 bp from the PAM. In contrast to SpCas9, FnCas9 activity fluctuates across genomic regions. Cotargeting dead Cas9 at proximal locations may alter local chromatin structures and render otherwise inaccessible target sites accessible and cleavable by FnCas9.