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Making ends meet: targeted integration of DNA fragments by genome editing

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Abstract

Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30–40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40–50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.

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Abbreviations

AAV:

Adeno-associated viral vector

a-NHEJ:

Alternative nonhomologous end-joining

Cas:

CRISPR-associated protein

c-NHEJ:

Canonical nonhomologous end-joining

CRISPR:

Clustered regularly interspaced short palindromic repeat

crRNA:

CRISPR RNA (the guide RNA)

ctRNP:

a complex of pre-assembled crRNA + tracRNA + Cas9 protein

dCas9:

catalytically inactive Cas9

DSB:

Double-strand break

dsDNA:

Double-stranded DNA

Easi-CRISPR:

Efficient additions with ssDNA inserts-CRISPR

HITI:

Homology-independent targeted integration

HDR:

Homology-directed repair

HMEJ:

Homology-mediated end-joining

HR:

Homologous recombination

Indels:

Small insertions and deletions

MMEJ:

Microhomology-mediated end-joining

NHEJ:

Nonhomologous end-joining

ObLiGaRe:

Obligate ligation-gated recombination.

PAM:

Protospacer-adjacent motif

PITCh:

Precise integration into target chromosome

SDSA:

DNA synthesis-dependent strand annealing

sgRNA:

single guide RNA containing a crRNA sequence and tracRNA (for CRISPR-Cas)

sgRNP:

complex of pre-assembled guide RNA and Cas9 protein

SSA:

Single-strand annealing

ssDNA:

Single-stranded DNA

ssODN:

Single-strand oligodeoxynucleotide

TALEN:

Transcription activator-like effector nuclease

tracrRNA:

Transactivating CRISPR RNA (a structural RNA to recruit Cas9)

2H2OP:

Two-hit by sgRNA and two oligos with a targeting plasmid

3H2OP:

Three-hit by sgRNA and two oligos with a targeting plasmid

ZFN:

Zinc finger nuclease

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Acknowledgements

Research in the author’s lab is supported by NIH R01 HG008160, NIH R01 GM121455, and NSF MCB 1607411.

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  1. Department of Molecular Biology, Cell Biology and Biochemistry, Brown University Division of Biology and Medicine, Sidney Frank Hall room 260, 185 Meeting Street, Providence, RI, 02912, USA

    Yutaka Yamamoto & Susan A. Gerbi

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  1. Yutaka Yamamoto
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Correspondence to Susan A. Gerbi.

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Yamamoto, Y., Gerbi, S.A. Making ends meet: targeted integration of DNA fragments by genome editing. Chromosoma 127, 405–420 (2018). https://doi.org/10.1007/s00412-018-0677-6

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