Journal
MOLECULAR BIOLOGY OF THE CELL
Volume 28, Issue 7, Pages 898-906Publisher
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E17-01-0051
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Funding
- Ministry of Education, Culture, Sports, Science and Technology, Japan [25113514, 15H01211]
- Japan Society for the Promotion of Science [22390013, 15H04370, 25860044, 15K07929]
- Uehara Memorial Foundation
- Astellas Foundation for Research on Metabolic Disorders
- Takeda Science Foundation
- JSPS Research Fellowship for Young Scientists
- Grants-in-Aid for Scientific Research [22390013, 15K07929, 15H01211, 25860044, 15H04370, 25113514, 15K14456] Funding Source: KAKEN
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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.
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