The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones.
BackgroundThe ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro.MethodsEach cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + .ResultsWe found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction.ConclusionsIn this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.
Homologous recombination is a technique used for performing precise genomic manipulations, and this makes it potentially ideal for gene therapy. The rate of spontaneous homologous recombination in human cells has been too low to be used experimentally or therapeutically but, by inducing a DNA double-strand break (DSB) in the target gene this rate can be stimulated. Zinc finger nucleases (ZFNs) are synthetic fusion proteins that can induce DSBs at specific sequences of DNA and stimulate gene targeting. Although the success of ZFNs in this application has been demonstrated, several issues remain. First, an optimal, generalized method of making effective and safe ZFNs needs to be determined. Second, a systematic method of evaluating the efficiency and safety of ZFNs is needed. We compared the gene-targeting efficiencies and cytotoxicity of ZFNs made using modular-assembly and ZFNs made using a bacterial 2-hybrid (B2H) selection-based method, in each case targeting the same single site. We found that a ZFN pair made using the B2H strategy is more efficient at stimulating gene targeting and less toxic than a pair made using modular-assembly. We demonstrate that a pair of three-finger B2H ZFNs is as efficient at stimulating gene targeting as ZFNs with more fingers, and induce similar or lower rates of toxicity.
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