Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Foundation, Tokyo, Japan.
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
J Gene Med. 2019 Nov;21(11):e3115. doi: 10.1002/jgm.3115. Epub 2019 Oct 29.
Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.
Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection.
We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites.
We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.
CRISPR/Cas9 系统的基因组编辑在基础研究中已有详细记录，预计将应用于基因治疗。同时表达多重向导 RNA（gRNA）和 Cas9/Cas9 衍生物对于高效敲除基因和安全的双切口策略很有吸引力。然而，由于通过同源重组缺失，高度多重 gRNA 表达单元很难在质粒中稳定维持，因此这种用途受到限制。
使用 lambda 体外包装代替转化来构建和制备大的、含有 cos 的质粒（cosmid）。使用四向导串联法获得含有多重 gRNA 单元的聚合酶链反应片段。通过脂质转染进行转染。
我们构建了新型 cosmid，其包含多达 16 个串联 gRNA 表达单元的线性化质粒-DNA 片段作为三聚体或四聚体（多聚体 cosmid）。这些 cosmid 的行为就像单体质粒一样，并且可以在没有缺失的情况下稳定地维持和扩增多重单元。令人惊讶的是，只需通过 lambda 包装扩增 cosmid 库，就可以去除缺失的 cosmid。含有多重 gRNA 单元和 Cas9 的 DNA 片段被转染到 293 细胞中，并发现通过删除预测位点之间的大片区来破坏乙型肝炎病毒的 X 基因。
我们提出了一种克服构建稳定含有多重 gRNA 表达单元的质粒的问题的简单方法。该方法可能使生产大量表达完整、高度多重 gRNA 和 Cas9/Cas9 衍生物的 DNA 片段成为可能，用于使用非病毒载体进行安全有效的基因组编辑治疗。
