Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.
Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.
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DOI：
10.1038/s41598-021-83259-0
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发表时间：
2021-02-17
影响因子：
4.6
通讯作者：
Saito I
中科院分区：
文献类型：
--
作者：
Nakanishi T;Maekawa A;Suzuki M;Tabata H;Sato K;Mori M;Saito I
多重引导RNA(GRNAs)的同时表达对于多个基因的敲除和通过引入多个缺失来有效干扰一个基因具有重要的意义。我们开发了一种利用lambda体外包装一步构建包含四个和八个多重gRNA表达单位的粘粒的四链导联串联方法。利用这种方法，我们制备了一个包含四个复合gRNA单元的腺病毒载体(ADV)，用于两个双划痕。出乎意料的是，ADV可以稳定地扩增到足以进行动物实验的规模，而且没有检测到缺乏多重单位。当含有针对H2-AA基因的gRNA的重组腺病毒与表达Cas9尼克酶的重组腺病毒混合感染小鼠胚胎成纤维细胞时，即使采用双划伤策略，也可以观察到80%以上的目的基因缺失。通过静脉注射，在新生小鼠肝细胞的两个位置也检测到约20%的靶基因中存在Indels。有趣的是，当一个双刻点被打乱时，另一个同时被打乱，这意味着在ADV系统中，同一细胞中的两个基因可能同时被打乱。表达4个复合gRNAs的ADV可同时敲除4个或2个基因，并具有较低的脱靶效应。
Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.
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影响因子：
4.6
作者：
Pei, Zheng;Shi, Guoli;Kondo, Saki;Ito, Masahiko;Maekawa, Aya;Suzuki, Mariko;Saito, Izumu;Suzuki, Tetsuro;Kanegae, Yumi
通讯作者：
Kanegae, Yumi
影响因子：
14.9
作者：
Ho TT;Zhou N;Huang J;Koirala P;Xu M;Fung R;Wu F;Mo YY
通讯作者：
Mo YY
编码CRISPR/CAS9的腺病毒载体在未选择的DMD肌肉细胞中挽救肌营养不良蛋白的合成。
影响因子：
4.6
作者：
Maggio I;Liu J;Janssen JM;Chen X;Gonçalves MA
通讯作者：
Gonçalves MA
影响因子：
4.2
作者：
LI, QT;KAY, MA;WOO, SLC
通讯作者：
WOO, SLC
影响因子：
7
作者：
Kim S;Kim D;Cho SW;Kim J;Kim JS
通讯作者：
Kim JS