Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system.
Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system.
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DOI：
10.1091/mbc.e17-01-0051
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发表时间：
2017-04-01
影响因子：
3.3
通讯作者：
Nakayama K
中科院分区：
文献类型：
--
作者：
Katoh Y;Michisaka S;Nozaki S;Funabashi T;Hirano T;Takei R;Nakayama K
构建了针对CRISPR/Cas9系统优化的供体敲入载体，并开发了一种实用的系统，该系统能够通过利用同源性非依赖性修复来有效破坏纤毛相关基因。该系统的第二个版本可用于降低脱靶切割频率并增加多功能性。CRISPR/Cas9系统彻底改变了几乎所有生物体的基因组编辑。尽管CRISPR/Cas9系统能够靶向切割基因组DNA，但其用于基因敲入仍然具有挑战性，因为同源重组活性的水平在各种细胞中不同。相比之下，同源非依赖性DNA修复的效率在大多数细胞类型中相对较高。因此，使用同源非依赖性修复机制是有效基因组编辑的可能替代方案。在这里，我们构建了一个针对CRISPR/Cas9系统优化的供体敲入载体，并开发了一个实用的系统，该系统能够通过利用同源性非依赖性修复来有效破坏靶基因。使用这种实用的敲入系统，我们成功地破坏了编码参与纤毛蛋白运输的蛋白质的基因，包括在hTERT-RPE 1细胞中具有低同源重组活性的IFT 88和IFT 20。使用CRISPR/Cas9系统最关键的问题是脱靶切割。为了降低脱靶切割频率并增加我们的敲入系统的通用性，我们构建了通用供体载体和含有具有增强的特异性和串联sgRNA表达盒的Cas9的表达载体。我们证明了我们系统的第二个版本提高了可用性。
A donor knock-in vector optimized for the CRISPR/Cas9 system is constructed and a practical system developed that enables efficient disruption of cilia-related genes by exploiting homology-independent repair. A second version of the system can be used to reduce off-target cleavage frequency and increase versatility. The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.