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Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV

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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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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438, P. R. China

    Qing Xiao, Taishan Min, Shuangping Ma, Lingna Hu, Hongyan Chen & Daru Lu

Authors
  1. Qing Xiao
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  2. Taishan Min
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  3. Shuangping Ma
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  4. Lingna Hu
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  5. Hongyan Chen
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  6. Daru Lu
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Contributions

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.

Corresponding authors

Correspondence to Hongyan Chen or Daru Lu.

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Conflict of interest

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.

Ethical approval

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.

Additional information

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

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