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FNCAS9P

Sigma-Aldrich

FnCas9 plasmid

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recombinant

expressed in E. coli

packaging

vial of 50 μL

concentration

20 ng/μL in TE buffer; DNA (1μg of plasmid DNA)

application(s)

CRISPR

Promoter

Promoter name: CMV

selection

kanamycin

shipped in

dry ice

storage temp.

−20°C

Related Categories

General description

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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Application

Functional Genomics/Target Validation
  • Proxy CRISPR
  • Creation of gene knockouts in multiple cell lines

Features and Benefits

  • Highly specific and highly active
  • Sequence verified
  • Ready to use purified plasmid DNA

Principle

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.

Legal Information

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 2

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable


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25G
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Structure and Engineering of Francisella novicida Cas9
Hirano, H. et al.
Cell, 164, 950-961 (2016)
Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.
Chen, F. et al.
Nature Communications, 8 (2017)

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