Skip to main content
Premium Trial:

Request an Annual Quote

Engineered Zinc Finger Nucleases Rapidly Generate Knockouts, Sangamo Says

NEW YORK (GenomeWeb News) – New research suggests Sangamo BioSciences’ zinc finger DNA-binding nucleases are an effective way to rapidly generate targeted gene knockouts in mammalian cell lines. Sangamo and Sigma-Aldrich touted the publication of the results today.
 
Scientists from Sangamo and Pfizer demonstrated the use of zinc finger nucleases for knocking out and/or mutating a specific gene — that coding for dihydrofolate reductase — in a Chinese hamster ovary mammalian cell line. Their results appeared online in the Proceedings of the National Academy of Sciences on March 21.
 
Although gene knockouts are a valuable tool for assessing gene function, creating engineered cell lines, and screening newly developed drugs, methods for quickly and reliably knocking out genes in mammalian cell lines remain elusive. Previous approaches have included chemical and ionizing mutagenesis and homologous recombination. Many researchers also utilize methods such as RNAi and other techniques to knock down gene expression without knocking out the gene.
 
For this study, the researchers exploited an imperfect and random repair mechanism called non-homologous end joining, which uses exonuclease activity to cut out additional base pairs before adding others and rejoining the DNA, rather than homology-directed repair, which fixes damaged DNA faithfully based on sister chromatid sequence.
 
To do this, they employed zinc-finger nucleases consisting of a targeted DNA-binding domain and the catalytic domain of an endonuclease enzyme called FokI. This enzyme can bind to and cut DNA, but only when it dimerizes or joins with a second piece of FokI. By designing ZFN pairs, researchers targeted specific stretches of DNA.
 
Using transiently expressed, site-specific zinc-finger nucleases, the team made double-strand DNA breaks in Exon 1 of the dihydrofolate reductase gene in a diploid Chinese hamster ovary cell line. They also compared the results using two different FokI domains: wild type or high-fidelity “obligate heterodimer” variants.
 
Five isolates — seven percent of the 68 generated with the ZFNs containing wild type FokI domains — contained mutated DHFR alleles. Of these, three were heterozygous DHFR disruptions and two appeared to be biallellic disruptions.
 
When they transfected the CHO-S cells with the FokI variant domains, the researchers found eleven clones — about three percent of those generated — that had at least one disrupted copy of DHFR, though most were small mutations of less than twenty base pairs. One clone, though, had a 302-base-pair mutation in one allele and a 38-base-pair mutation in the other.
 
Subsequent experiments, including Western blot analyses and functional analyses, suggest that the three DHFR “knockout” cell lines did not produce functional dihydrofolate reductase.
 
Overall, the frequency of mutated alleles was roughly two to three percent. Still, with more than one percent showing biallelic modification, the authors argue that “ZFN-mediated biallelic disruption at a specific locus — in the absence of selection — offers a significant advantage compared with existing methods.” The targeted gene disruption reportedly takes two or three days, while the entire process takes about a month.
 
“The precise nature of the mutations generated by [non-homologous end joining]-based repair of the ZFN-induced DSB cannot be predetermined and, indeed, need not be known,” the authors wrote. “[W]e show that the frequency of gene-disrupting mutations generated by this stochastic process is more than sufficient for utility as a method for gene knockout.”
 
Sangamo President and CEO Edward Lanphier said in a statement that the company intends to file Investigative New Drug Applications this year to test HIV/AIDS and brain cancer treatments developed by knocking out key genes involved in each condition.
 
Sigma-Aldrich is the exclusive licensee of Sangamo’s ZFN technology in the research reagent field.

The Scan

Highly Similar

Researchers have uncovered bat viruses that are highly similar to SARS-CoV-2, according to Nature News.

Gain of Oversight

According to the Wall Street Journal, the Biden Administration is considering greater oversight of gain-of-function research.

Lasker for mRNA Vaccine Work

The Scientist reports that researchers whose work enabled the development of mRNA-based vaccines are among this year's Lasker Award winners

PLOS Papers on Causal Variant Mapping, Ancient Salmonella, ALK Fusion Test for NSCLC

In PLOS this week: MsCAVIAR approach to map causal variants, analysis of ancient Salmonella, and more.