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Life Science > Zinc Finger Nuclease (ZFN) > CompoZr® Knockout ZFNs
Zinc Finger Nuclease (ZFN)

CompoZr® Knockout ZFNs

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Ready-to-use, pre-validated ZFNs for permanent knockout of your favorite gene

Targeted gene knockout made easy
CompoZr Zinc Finger Nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate highly specific targeted genome editing by creating a single double-strand break in the target gene in a sequence-specific manner. Sigma-Aldrich® now offers ready-to-use pre-constructed and validated CompoZr Knockout ZFNs that are designed to permanently knockout your gene of interest in a quick, simple and target specific manner. The Knockout ZFNs are designed to a sequence within the first two-thirds of the gene target, ensuring that the gene disruption will lead to a knockout. Simply choose one of the pre-validated CompoZr Knockout ZFN pairs by browsing through the list of validated ZFNs. 

Benefits

Knockout ZFNs will ship within 3 weeks of order

  • Knockout ZFNs are already designed, pre-constructed and pre-validated

Ready-to-use

  • Simply transfect the pre-validated ZFNs against your gene of interest into your cell line
  • Screen for gene disruption in as little as 48 hours
  • Single cell clone out individual cells with gene disruptions

Knockouts induced through a single transfection experiment

  • Single or biallelic edits occur in 1-20% of clone population
  • No antibiotic selection required for screening

High specificity

  • CompoZr ZFN technology provides for targeted single locus gene modification
  • ZFN activity at non-specific sites are negligible (see references)
  • Genes are permanently disrupted at the target site and remain heritable


CompoZr® Knockout ZFNsFigure 1: CompoZr Knockout ZFNs

(A) Ready-to-transfect Zinc Finger Nuclease
ZFN pairs are provided as ready-to-transfect plasmid DNA and mRNA, for the cell line of your choice. Each format can be transfected into cells to be transcribed into the ZFN protein. The ZFN binds a specific DNA sequence within the target gene, inducing a double strand break at the bound site. This break is repaired by an imperfect endogenous repair mechanism called non-homologous end joining (NHEJ). Due to the imperfect nature of NHEJ, a percentage of double-strand breaks within a ZFN-treated cellular population will be misrepaired by the addition and/or deletion of bases. These disruptions within the gene occur at the site of the break resulting in a non-sense gene product, the loss of gene function and a knockout. CompoZr Knockout ZFNs deliver highly successful gene knockout frequencies of up to 20% in several cell lines, without the need for any selection markers.

 

(B) ZFN-induced double strand break
The transfected ZFNs bind their target DNA and induce a double strand break. The double strand break is repaired via NHEJ. The imperfect NHEJ causes mutations to be incorporated within the specific gene.

 

(C) Cel1 Assay
A nucleotide mismatch assay is used to detect the presence of mutant clones and quantify the effectiveness of ZFNs. The mismatch assay consists of amplifying the target region from ZFN-treated genomic DNA via standard PCR using the primers provided in the kit. Resultant PCR products are denatured and allowed to re-anneal. A fraction of re-annealed products will contain bulges of DNA where heterogeneous mismatches occur. When a mismatch sensitive enzyme is added to the reaction, DNA digestion will occur at the site of the nucleotide mismatch. PCR pools are analyzed via PAGE. Lanes 2 and 3 contain cells that have been edited via ZFN-induced DSBs. The presence of digested PCR product indicates ZFN-mediated mutagenesis and the ratio of digested PCR products to wildtype PCR product (quantified by densitometry) determines ZFN activity.

 



See a complete list of CompoZr Knockout ZFNs
CompoZr® ZFN Add Delete Swap



ZFN Knockout Kit components

  • 10 aliquots of ready-to-deliver mRNA
  • Plasmid DNA for each ZFN in the pair
  • Forward and reverse primers for screening the DNA locus for mutation
  • Genomic DNA as a positive control for PCR (from validated samples where the target gene has been mutated using the supplied CompoZr Knockout ZFN)

All Zinc Finger Nucleases are sold under license from Sangamo Biosciences. For a copy of the Label License provided with purchase of CompoZr ZFNs, please visit the ZFN Label License page. CompoZr is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.

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