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    Home > Food News > Food Articles > CRISPR/Cas9-mediated non-isoid DNA knocks into the application of multi-copy gene knock-| BMC Biology

    CRISPR/Cas9-mediated non-isoid DNA knocks into the application of multi-copy gene knock-| BMC Biology

    • Last Update: 2021-03-14
    • Source: Internet
    • Author: User
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    Title: Homology-independent multiallelic disruption via CRISPR/Cas9-base knock-in-yields distinct outcomes in human cells
    Journal:
    Chenzi Zhang†, Xiangjun He†, Yvonne K. Kwok, Feng Wang, Junyi Xue, Hui Zhao, Kin Wah Suen, Chi Chiu Wang, Jianwei Ren, George G. Chen, Paul B. S. Lai, Jiangchao Lichao, Yin Xia, Andrew M. Chan, Wai-Yee Chan and Feng bo
    Published: 2018/12/28
    Digital Identification Number: 10.1186/s12915-018-0616-2
    Original Link:
    WeChat Link: in
    Biomedical Research, in-body culture of various cell line is an important model for the study of human gene function. However, these cell lineages often accumulate and carry a large number of genomic abnormalities during screening to establish and adapt to culture conditions, with many genes presented as three copies or more (Figure 1). Therefore, in the course of research, it is often difficult to edit and completely eliminate multi-copy genes in introphy cell line.previous studies have found that DNA repair induced by CRISPR/Cas9, mediated by non-homogeneity end connection paths (non-homology end-joining, NHEJ pathway), can be used to target large pieces of DNA (or genes) in the genome. The knock-in efficiency of this method is much higher than that of traditional knock-ins mediated by the homology-dependent pathway (homology-dependent repair, HDR pathway) (He et al., Nucleic Acids Research 2016; Suzuki et al., Nature 2016; Zhou et al., FEBS Letters 2016)。 Therefore, this method may provide a good tool for multi-copy gene knock-out.
    Recently, a new study published in
    by Dr. Feng Bo of the School of Biomedical Sciences of Chinese University of Hong Kong, modeled on LO2 human cell lineages, using CRISPR/Cas9-NHEJ-mediated methods, inserted two fluorescent reporting genes (ires-GFP/ires-Tdomato) into the target gene at the same time. These reporting genes can only be expressed by the destination gene launcher after the intended gene has been inserted in a pre-designed manner. Subsequently, the sample can be detected and sorted to obtain double-positive fluorescent cells through fluorescence fluorescence sorting technology (Fluorescence activated cell sorting, FACS), which can be achieved in one step by blocking the expression of two or more copies of genes at the same time. Using this method, they performed gene knock-out experiments and tests on the candidate genes ULK1 (4 copies) and FAT10 (3 copies) in LO2 (Figure 2).In the analysis of monoclonal cell line, genomic PCR verified that the corresponding fluorescent reporting gene was inserted at the shear points of the ULK1 and FAT10 genes, and mRNA, protein level and functional analysis verified the absence of ULK1 and FAT10 proteins in single cloned cell line (in the case of ULK1 monoclonal cells, Figure 3). Moreover, the overall experimental results show that the method can indeed significantly improve the efficiency of obtaining monoclonal cell lineages that carry the gene of interest (sic) (sic) 2D)。At the same time, the study also used the same method to insert double fluorescence-reporting genes into the CTIP gene (2 copies; Figure 1) exons, and unexpectedly found that ctIP transcription and protein expression could still be detected at the mRNA and protein levels, although the CTIP double-allied gene was clearly detected in the obtained monoclonal cell line.
    further sequencing of CTIP mRNAs in these cloned cell lineages showed that the mRNA transcripts did come from the genome after the reported gene had been inserted and carried a variety of shear mark. Surprisingly, however, all of these abnormal CTIP mRNA transcripts retain the reading framework of the original CTIP gene (in-frame aberrant transcripts), (Figure 4).This study used CRISPR/Cas9-induced NHEJ repair to insert dual fluorescent reporting genes to achieve the knock-out of multi-copy genes in introphy cell line. This method can use dual fluorescent streaming to collect multi-copy gene knock-out cells, simplify and improve the efficiency of multi-copy gene knock-out.
    In addition, by editing the genes of CTIP, this study found that a variety of DNA repair and RNA shear mechanisms present in cells produce a variety of random products, which give cells the opportunity to collect functional gene products under survival pressure, so that the necessary genes that play a key role in cell survival are not completely "knocked out".
    study further deepens the understanding of cellular responses in gene editing, and provides important guidance for the future application of CRISPR/Cas9 gene editing.
    Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1−/− cells and attenuated cytokine-induced cell death in FAT10−/− clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIPtranscripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIPtranscripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.(
    , 5.770 -
    , 7.556 -
    ) is an open access journal publishing outstanding research in all areas of biology, with a publication policy that combines selection for broad interest and importance with a commitment to serving authors well.
    (Source: Science.com)
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