Abstract
Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13–14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6–19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.
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Abbreviations
- CRISPR/Cas9:
-
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease
- MMEJ:
-
Microhomology-mediated end-joining
- ssDNA:
-
Single-strand DNA
- dsDNA:
-
Double-strand DNA
- ITR:
-
Inverted terminal repeat
- AAV:
-
Adeno-associated virus
- gRNA:
-
Guide RNA
- DSB:
-
Double-strand break
- HDR:
-
Homology-directed repair
- NHEJ:
-
Non-homologous end joining
- HA:
-
Homology arm
- PITCH:
-
Precise integration into target chromosome
- ssODN:
-
Single-strand oligodeoxynucleotide
- HR:
-
Homologous recombination
- HP:
-
Hairpin
- PAM:
-
Protospacer adjacent motif
- SNP:
-
Single nucleotide polymorphism
- RNP:
-
Ribonucleoprotein
- HEK:
-
Human embryonic kidney
- PEI:
-
Polyethylenimine
- qRT-PCR:
-
Quantitative real-time PCR
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- FBS:
-
Fetal bovine serum
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Acknowledgements
We are grateful to Prof. Hui Yang for kindness gift of Cas9 expressing vector and MMEJ plasmid, to Dr. Cong Huai and Mr. Chenqiang Jia for their help in language polishing, to Mr. Hairui Xi and Mr. Renji Zhang for providing technical assistance.
Funding
This study was funded by the National Natural Science Foundation of China (Grant Nos. 81772657, 31571371 and 81372706) and Precision Medicine Research Key Project of National Key R&D Plan (Grant No. 2017YFC0907501).
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HC and DL conceptualized and designed the project. QX and TM performed all the experiments. QX and LH analyzed and interpreted the data. QX and SM wrote the manuscript, which all authors edited and approved.
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Qing Xiao declares that she has no conflict of interest. Taishan Min declares that she has no conflict of interest. Shuangping Ma declares that she has no conflict of interest. Lingna Hu declares that she has no conflict of interest. Hongyan Chen declares that she has no conflict of interest. Daru Lu declares that he has no conflict of interest.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Communicated by S. Hohmann.
Hongyan Chen and Daru Lu are co-corresponding authors.
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Xiao, Q., Min, T., Ma, S. et al. Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV. Mol Genet Genomics 293, 1051–1060 (2018). https://doi.org/10.1007/s00438-018-1437-2
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DOI: https://doi.org/10.1007/s00438-018-1437-2