Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure
Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure
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DOI：
10.1002/jgm.3115
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发表时间：
2019-10-29
影响因子：
3.5
通讯作者：
Saito, Izumu
中科院分区：
文献类型：
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作者：
Nakanishi, Tomoko;Maekawa, Aya;Saito, Izumu
背景使用CRISPR/CAS9系统的基因组编辑现已在基础研究中得到很好的证明，并有望应用于基因治疗。多重引导RNA(GRNA)和Cas9/Cas9衍生物的同时表达具有高效的基因敲除和安全的双缺口策略。然而，这种用途是有限的，因为由于同源重组的缺失，高效表达gRNA的单位很难稳定地保持在质粒中。方法用Lambda体外包装代替转化法构建和制备含cos的大质粒(粘粒)。用四导联法获得含有多个gRNA单位的聚合酶链式反应片段。通过脂质体转染法进行转染法。结果我们构建了由线性化的质粒DNA片段组成的新型粘粒，其中含有多达16个拷贝的多重gRNA表达单位作为三聚体或四聚体(多边形粘粒)。这些粘粒的行为就像它们是单体质粒一样，并且多重单位可以稳定地保持和扩增，而不存在缺失。令人惊讶的是，删除的粘粒仅仅是通过使用lambda包装放大粘粒来移除的。将含有多个gRNA单位和Cas9的DNA片段导入293细胞，发现通过删除预测位点之间的一大段区域来干扰乙肝病毒的X基因。结论我们提出了一种简单的方法来克服稳定构建含有多个gRNA表达单位的质粒的问题。该方法可以生产大量表达完整的、高度多元化的gRNA和Cas9/Cas9衍生物的DNA片段，用于使用非病毒载体进行安全和有效的基因组编辑治疗。
Background 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. Methods 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. Results 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. Conclusions 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.