PUBLICATION

Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species

Authors
He, X., Chen, W., Liu, Z., Yu, G., Chen, Y., Cai, Y.J., Sun, L., Xu, W., Zhong, L., Gao, C., Chen, J., Zhang, M., Yang, S., Yao, Y., Zhang, Z., Ma, F., Zhang, C.C., Lu, H.P., Yu, B., Cheng, T.L., Qiu, J., Sheng, Q., Zhou, H.M., Lv, Z.R., Yan, J., Zhou, Y., Qiu, Z., Cui, Z., Zhang, X., Meng, A., Sun, Q., Yang, Y.
ID
ZDB-PUB-200403-223
Date
2020
Source
Nucleic acids research   48(10): e57 (Journal)
Registered Authors
Meng, Anming
Keywords
none
MeSH Terms
  • Humans
  • Mice
  • DNA-Binding Proteins/metabolism
  • Animals
  • Gene Knock-In Techniques
  • Genomics
  • Homologous Recombination
  • Macaca fascicularis
  • Rec A Recombinases/metabolism
  • Bacterial Proteins/metabolism*
  • Rats, Sprague-Dawley
  • Zebrafish/genetics
  • Female
  • DNA Breaks, Double-Stranded
  • Gene Editing/methods*
  • INDEL Mutation
(all 16)
PubMed
32232370 Full text @ Nucleic Acids Res.
Abstract
Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.
Genes / Markers
Figures
Figure Gallery (6 images)
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Expression
Phenotype
No data available
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
zf3702TgTransgenic Insertion
zf3703TgTransgenic Insertion
zf3704TgTransgenic Insertion
zf3705TgTransgenic Insertion
1 - 4 of 4
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Human Disease / Model
No data available
Sequence Targeting Reagents
Target Reagent Reagent Type
bmp2bCRISPR2-bmp2bCRISPR
bmp2bCRISPR3-bmp2bCRISPR
gfapCRISPR4-gfapCRISPR
gfapCRISPR5-gfapCRISPR
gfapCRISPR6-gfapCRISPR
lft2CRISPR1-lft2CRISPR
ndr2CRISPR21-ndr2CRISPR
ndr2CRISPR22-ndr2CRISPR
1 - 8 of 8
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Fish
No data available
Antibodies
No data available
Orthology
No data available
Engineered Foreign Genes
Marker Marker Type Name
ChR2EFGChR2
Dendra2EFGDendra2
EYFPEFGEYFP
1 - 3 of 3
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Mapping
No data available
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Citation
He, X., Chen, W., Liu, Z., Yu, G., Chen, Y., Cai, Y.J., Sun, L., Xu, W., Zhong, L., Gao, C., Chen, J., Zhang, M., Yang, S., Yao, Y., Zhang, Z., Ma, F., Zhang, C.C., Lu, H.P., Yu, B., Cheng, T.L., Qiu, J., Sheng, Q., Zhou, H.M., Lv, Z.R., Yan, J., Zhou, Y., Qiu, Z., Cui, Z., Zhang, X., Meng, A., Sun, Q., Yang, Y. (2020) Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species. Nucleic acids research. 48(10):e57.