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November 15, 2011 (Vol. 31, No. 20)

Advances in Sophisticated Genome Editing

ssDNA Oligos and CompoZr ZFNs More Efficiently Produce Nucleotide Insertions and/or Deletions

  • Results

    Click Image To Enlarge +
    Figure 2. (A) 3% agarose gel displaying desired biallelic BamHI mutation in 20 clones. (B) Immunoblots probing the sensitivity of wild-type and RSK2-C436V modified cells to the small molecule RSK kinase inhibitor fmk. Three independent human K562 clones were tested.

    Using the RSK2 125 mer ssODN and RSK2 ZFNs, BamHI conversion rates of 22–32% were detected by the RFLP assay (Figure 1B). Single-cell cloning yielded at least 20 clones from 750 with a biallelic BamHI conversion (Figure 2A) and the desired cysteine to valine codon modification (DNA sequencing data not shown). Immunoblotting of three randomly selected mutants demonstrated the predicted insensitivity of RSK2 to the RSK kinase inhibitor fmk (Figure 2B).

  • Discussion

    As a method for characterization of eukaryotic proteins, codon-based mutagenesis has been restricted to cDNAs harbored on exogenous plasmids. In this application, the coding sequence for the RSK2 kinase was modified at the endogenous location on human chromosome X in the context of: (1) the native promoter and all associated 5´-UTR regulatory elements, (2) intron/exon splicing, (3) mRNA export and processing, and (4) any 3´-UTR regulatory elements such as microRNA binding sites. This ensures that the expression levels of the RSK2 kinase and associated small molecule kinetics are controlled in the truest biological fashion.

    Previously, this mode of endogenous protein manipulation was limited to certain model bacteria and yeast. Now, it is a realistic experimental option for mammalian cells using ZFNs in combination with ssODN or plasmid donors.

    A major technical challenge for ZFN-based genome editing has been the engineering of ZFNs within close range (<200 bp) of the desired mutation site. The use of ssODNs layers an even tighter restriction on ZFN design resolution since ssODN length is often limited to <130 bp for economical and quality reasons.

    Any genome-editing method must achieve a high level of accuracy to be useful. CompoZr ZFNs utilize a multifaceted approach that includes bioinformatics, FokI nuclease engineering, and a proprietary library of ZFN modules to assure specificity. Bioinformatics is used to avoid homologous genomic regions, SNPs, splice variants, and repeat elements.

    Also, nearly all CompoZr ZFNs have target sites at least 30 bp long that differ from any other site in the genome of interest by at least four nucleotides. These bioinformatic steps assure specificity and are applied to all CompoZr-engineered ZFNs, both Custom ZFNs and readily available human, rat, or mouse gene Knockout ZFNs.

    In addition to these strict bioinformatic measures, CompoZr ZFN’s FokI domain requires heterodimerization to create a DSB, providing a significantly higher level of specificity for all CompoZr ZFNs regardless of the genetic locus being studied.

    Combining the use of ssODN templates and exacting measures to ensure the specificity of CompoZr ZFNs maximizes the efficiency and subsequent utility of CompoZr ZFN-mediated genome editing.

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