Nature Biotechnology, volume 36, issue 7, pages 632-637
Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos
1
Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada
|
2
Department of Molecular Genetics, University of Toronto, Toronto, Canada
|
Publication type: Journal Article
Publication date: 2018-06-11
Journal:
Nature Biotechnology
Quartile SCImago
Q1
Quartile WOS
Q1
Impact factor: 46.9
ISSN: 10870156, 15461696
DOI:
10.1038/nbt.4166
Molecular Medicine
Applied Microbiology and Biotechnology
Biotechnology
Bioengineering
Biomedical Engineering
Abstract
Knock-in mice with precise insertions are efficiently generated through delivery of CRISPR–Cas9 to two-cell embryos. Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, we describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin–streptavidin approach to localize repair templates to target sites, we achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. We targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. We also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. We suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.
Citations by journals
|
2
4
6
8
10
|
|
|
Nature Communications
|
Nature Communications
10 publications, 4.57%
|
|
Methods in Molecular Biology
|
Methods in Molecular Biology
8 publications, 3.65%
|
|
Scientific Reports
|
Scientific Reports
7 publications, 3.2%
|
|
International Journal of Molecular Sciences
|
International Journal of Molecular Sciences
6 publications, 2.74%
|
|
Development (Cambridge)
|
Development (Cambridge)
5 publications, 2.28%
|
|
Genes
|
Genes
5 publications, 2.28%
|
|
Cell Reports
|
Cell Reports
5 publications, 2.28%
|
|
Methods
|
Methods
4 publications, 1.83%
|
|
Cell
|
Cell
4 publications, 1.83%
|
|
BMC Biotechnology
|
BMC Biotechnology
3 publications, 1.37%
|
|
Nucleic Acids Research
|
Nucleic Acids Research
3 publications, 1.37%
|
|
eLife
|
eLife
3 publications, 1.37%
|
|
Cold Spring Harbor Protocols
|
Cold Spring Harbor Protocols
3 publications, 1.37%
|
|
Molecular Biology Reports
|
Molecular Biology Reports
2 publications, 0.91%
|
|
Biology Open
|
Biology Open
2 publications, 0.91%
|
|
The CRISPR Journal
|
The CRISPR Journal
2 publications, 0.91%
|
|
Open Biology
|
Open Biology
2 publications, 0.91%
|
|
Frontiers in Genetics
|
Frontiers in Genetics
2 publications, 0.91%
|
|
Frontiers in Immunology
|
Frontiers in Immunology
2 publications, 0.91%
|
|
Nature Cell Biology
|
Nature Cell Biology
2 publications, 0.91%
|
|
Applied Microbiology and Biotechnology
|
Applied Microbiology and Biotechnology
2 publications, 0.91%
|
|
Current Opinion in Genetics and Development
|
Current Opinion in Genetics and Development
2 publications, 0.91%
|
|
Cell Stem Cell
|
Cell Stem Cell
2 publications, 0.91%
|
|
STAR Protocols
|
STAR Protocols
2 publications, 0.91%
|
|
PLoS Genetics
|
PLoS Genetics
2 publications, 0.91%
|
|
PLoS Biology
|
PLoS Biology
2 publications, 0.91%
|
|
Developmental Cell
|
Developmental Cell
2 publications, 0.91%
|
|
Molecular Therapy - Nucleic Acids
|
Molecular Therapy - Nucleic Acids
2 publications, 0.91%
|
|
Nanoscale
|
Nanoscale
2 publications, 0.91%
|
|
2
4
6
8
10
|
Citations by publishers
|
5
10
15
20
25
30
35
40
45
50
|
|
|
Springer Nature
|
Springer Nature
49 publications, 22.37%
|
|
Elsevier
|
Elsevier
42 publications, 19.18%
|
|
Multidisciplinary Digital Publishing Institute (MDPI)
|
Multidisciplinary Digital Publishing Institute (MDPI)
18 publications, 8.22%
|
|
Frontiers Media S.A.
|
Frontiers Media S.A.
9 publications, 4.11%
|
|
The Company of Biologists
|
The Company of Biologists
8 publications, 3.65%
|
|
Wiley
|
Wiley
6 publications, 2.74%
|
|
Public Library of Science (PLoS)
|
Public Library of Science (PLoS)
4 publications, 1.83%
|
|
Cold Spring Harbor Laboratory
|
Cold Spring Harbor Laboratory
4 publications, 1.83%
|
|
The Royal Society
|
The Royal Society
3 publications, 1.37%
|
|
Royal Society of Chemistry (RSC)
|
Royal Society of Chemistry (RSC)
3 publications, 1.37%
|
|
Oxford University Press
|
Oxford University Press
3 publications, 1.37%
|
|
eLife Sciences Publications
|
eLife Sciences Publications
3 publications, 1.37%
|
|
Mary Ann Liebert
|
Mary Ann Liebert
2 publications, 0.91%
|
|
Society for the Study of Reproduction
|
Society for the Study of Reproduction
2 publications, 0.91%
|
|
American Association for the Advancement of Science (AAAS)
|
American Association for the Advancement of Science (AAAS)
2 publications, 0.91%
|
|
Proceedings of the National Academy of Sciences (PNAS)
|
Proceedings of the National Academy of Sciences (PNAS)
2 publications, 0.91%
|
|
Rockefeller University Press
|
Rockefeller University Press
2 publications, 0.91%
|
|
PeerJ
|
PeerJ
1 publication, 0.46%
|
|
SAGE
|
SAGE
1 publication, 0.46%
|
|
Korean Society for Biotechnology and Bioengineering
|
Korean Society for Biotechnology and Bioengineering, 1, 0.46%
Korean Society for Biotechnology and Bioengineering
1 publication, 0.46%
|
|
Maad Rayan Publishing Company
|
Maad Rayan Publishing Company, 1, 0.46%
Maad Rayan Publishing Company
1 publication, 0.46%
|
|
American Society for Biochemistry and Molecular Biology
|
American Society for Biochemistry and Molecular Biology
1 publication, 0.46%
|
|
Biophysical Society
|
Biophysical Society
1 publication, 0.46%
|
|
IEEE
|
IEEE
1 publication, 0.46%
|
|
Genetics Society of America
|
Genetics Society of America
1 publication, 0.46%
|
|
Nanjing Medical University
|
Nanjing Medical University, 1, 0.46%
Nanjing Medical University
1 publication, 0.46%
|
|
Colegio Brasileiro de Reproducao Animal
|
Colegio Brasileiro de Reproducao Animal, 1, 0.46%
Colegio Brasileiro de Reproducao Animal
1 publication, 0.46%
|
|
Higher Education Press
|
Higher Education Press
1 publication, 0.46%
|
|
Bio-Protocol
|
Bio-Protocol, 1, 0.46%
Bio-Protocol
1 publication, 0.46%
|
|
5
10
15
20
25
30
35
40
45
50
|
- We do not take into account publications that without a DOI.
- Statistics recalculated only for publications connected to researchers, organizations and labs registered on the platform.
- Statistics recalculated weekly.
{"yearsCitations":{"type":"bar","data":{"show":true,"labels":[2018,2019,2020,2021,2022,2023,2024],"ids":[0,0,0,0,0,0,0],"codes":[0,0,0,0,0,0,0],"imageUrls":["","","","","","",""],"datasets":[{"label":"Citations number","data":[6,33,38,48,42,44,8],"backgroundColor":["#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6"],"percentage":["2.74","15.07","17.35","21.92","19.18","20.09","3.65"],"barThickness":null}]},"options":{"indexAxis":"x","maintainAspectRatio":true,"scales":{"y":{"ticks":{"precision":0,"autoSkip":false,"font":{"family":"Montserrat"},"color":"#000000"}},"x":{"ticks":{"stepSize":1,"precision":0,"font":{"family":"Montserrat"},"color":"#000000"}}},"plugins":{"legend":{"position":"top","labels":{"font":{"family":"Montserrat"},"color":"#000000"}},"title":{"display":true,"text":"Citations per year","font":{"size":24,"family":"Montserrat","weight":600},"color":"#000000"}}}},"journals":{"type":"bar","data":{"show":true,"labels":["Nature Communications","Methods in Molecular Biology","Scientific Reports","International Journal of Molecular Sciences","Development (Cambridge)","Genes","Cell Reports","Methods","Cell","BMC Biotechnology","Nucleic Acids Research","eLife","Cold Spring Harbor Protocols","Molecular Biology Reports","Biology Open","The CRISPR Journal","Open Biology","Frontiers in Genetics","Frontiers in Immunology","Nature Cell Biology","Applied Microbiology and Biotechnology","Current Opinion in Genetics and Development","Cell Stem Cell","STAR Protocols","PLoS Genetics","PLoS Biology","Developmental Cell","Molecular Therapy - Nucleic Acids","Nanoscale"],"ids":[3231,3516,13767,14627,2238,8879,9130,16671,16552,8791,23904,24138,24846,2791,24546,26834,13367,13242,1699,22534,17681,11302,13752,28368,785,13133,17069,20403,228],"codes":[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0],"imageUrls":["\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/ko5EaisKGm66J1hHXtfEgVEeeWb9uOcMofpNuf0a_medium.webp","\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/yNSijlgQghQF53VZuyFLA30CKDe4j3HK74Vtpnxa_medium.webp","\/storage\/images\/resized\/eIZgkq1MRZyJ3hBkdupnUUv7wgB6zCl9D99SlPN5_medium.webp","\/storage\/images\/resized\/hg4jJjT8wVGtHstBCc0zk465Mg9pLx3G4odCDOqE_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/ko5EaisKGm66J1hHXtfEgVEeeWb9uOcMofpNuf0a_medium.webp","\/storage\/images\/resized\/Zy5YST233rzRbZP24I24MzZm64hz20ZV9qQopmMn_medium.webp","\/storage\/images\/resized\/BHBStD1OkQqtKFRRjS5njgNe7bXCAEgaDQVK7GTA_medium.webp","\/storage\/images\/resized\/4QWA67eqfcfyOiA8Wk7YnqroHFqQbTsmDJUYTCTg_medium.webp","\/storage\/images\/resized\/4QWA67eqfcfyOiA8Wk7YnqroHFqQbTsmDJUYTCTg_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/PRGhlgB4OKRltSNtT39eA6wlnOTGRap1QQ6FQHih_medium.webp","\/storage\/images\/resized\/PRGhlgB4OKRltSNtT39eA6wlnOTGRap1QQ6FQHih_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/leiAYcRDGTSl5B1eCnwpSGqmDEUEfDPPoYisFGhT_medium.webp"],"datasets":[{"label":"","data":[10,8,7,6,5,5,5,4,4,3,3,3,3,2,2,2,2,2,2,2,2,2,2,2,2,2,2,2,2],"backgroundColor":["#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6"],"percentage":[4.57,3.65,3.2,2.74,2.28,2.28,2.28,1.83,1.83,1.37,1.37,1.37,1.37,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91,0.91],"barThickness":13}]},"options":{"indexAxis":"y","maintainAspectRatio":false,"scales":{"y":{"ticks":{"precision":0,"autoSkip":false,"font":{"family":"Montserrat"},"color":"#000000"}},"x":{"ticks":{"stepSize":null,"precision":0,"font":{"family":"Montserrat"},"color":"#000000"}}},"plugins":{"legend":{"position":"top","labels":{"font":{"family":"Montserrat"},"color":"#000000"}},"title":{"display":true,"text":"Journals","font":{"size":24,"family":"Montserrat","weight":600},"color":"#000000"}}}},"publishers":{"type":"bar","data":{"show":true,"labels":["Springer Nature","Elsevier","Multidisciplinary Digital Publishing Institute (MDPI)","Frontiers Media S.A.","The Company of Biologists","Wiley","Public Library of Science (PLoS)","Cold Spring Harbor Laboratory","The Royal Society","Royal Society of Chemistry (RSC)","Oxford University Press","eLife Sciences Publications","Mary Ann Liebert","Society for the Study of Reproduction","American Association for the Advancement of Science (AAAS)","Proceedings of the National Academy of Sciences (PNAS)","Rockefeller University Press","PeerJ","SAGE","Korean Society for Biotechnology and Bioengineering","Maad Rayan Publishing Company","American Society for Biochemistry and Molecular Biology","Biophysical Society","IEEE","Genetics Society of America","Nanjing Medical University","Colegio Brasileiro de Reproducao Animal","Higher Education Press","Bio-Protocol"],"ids":[8,17,202,208,872,11,344,6909,259,123,19,6627,230,2584,189,162,22,1089,31,2287,7180,2090,1454,6953,3356,1401,1444,321,7047],"codes":[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0],"imageUrls":["\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/4QWA67eqfcfyOiA8Wk7YnqroHFqQbTsmDJUYTCTg_medium.webp","\/storage\/images\/resized\/ko5EaisKGm66J1hHXtfEgVEeeWb9uOcMofpNuf0a_medium.webp","\/storage\/images\/resized\/bRyGpdm98BkAUYiK1YFNpl5Z7hPu6Gd87gbIeuG3_medium.webp","\/storage\/images\/resized\/PRGhlgB4OKRltSNtT39eA6wlnOTGRap1QQ6FQHih_medium.webp","\/storage\/images\/resized\/hg4jJjT8wVGtHstBCc0zk465Mg9pLx3G4odCDOqE_medium.webp","\/storage\/images\/resized\/BHBStD1OkQqtKFRRjS5njgNe7bXCAEgaDQVK7GTA_medium.webp","\/storage\/images\/resized\/leiAYcRDGTSl5B1eCnwpSGqmDEUEfDPPoYisFGhT_medium.webp","\/storage\/images\/resized\/yNSijlgQghQF53VZuyFLA30CKDe4j3HK74Vtpnxa_medium.webp","\/storage\/images\/resized\/eIZgkq1MRZyJ3hBkdupnUUv7wgB6zCl9D99SlPN5_medium.webp","\/storage\/images\/resized\/Zy5YST233rzRbZP24I24MzZm64hz20ZV9qQopmMn_medium.webp","\/storage\/images\/resized\/OQ7TJyrdS1q8HnKdPSYhEebWzI7q5LxLYiQeWuua_medium.webp","\/storage\/images\/resized\/s10mcsCV4OAUg9O2KrqOquQC0PhyLMI8hUUkuflM_medium.webp","\/storage\/images\/resized\/mxFdPe9qujsfvfYfcN0QOclAiYORFb0xrRlwV8gs_medium.webp","\/storage\/images\/resized\/lEA1FyUnFAZR1DchuczZi5bjAixG3TNeWVz6oF4R_medium.webp","\/storage\/images\/resized\/aOPqEqNWK2Sq9T8CLDlsR6diWsFkHppx3bLsAnpx_medium.webp","\/storage\/images\/resized\/ruydfaB80LDjlkYqsfOeUAZohOIODyq7bQzis5O7_medium.webp","","","\/storage\/images\/resized\/OuA6WPZekwEiUM99mP2pSqj4yHPxHezM6xgLhAjO_medium.webp","\/storage\/images\/resized\/Bmz9XNb2QDptmPTa5N8huM54tShVewUj8fZqKFRy_medium.webp","\/storage\/images\/resized\/6scCJegesojp2jubwY3uKCzTAmgsaH2GIFlg6Hfk_medium.webp","\/storage\/images\/resized\/9EVpRifnnUJt6oHGY5GfsfSXTEz5wzbdgnZqiF5m_medium.webp","","","\/storage\/images\/resized\/fzjeYAS7sc3p4k3vIdQIac4L4J6MUyfSbYzJSIAv_medium.webp",""],"datasets":[{"label":"","data":[49,42,18,9,8,6,4,4,3,3,3,3,2,2,2,2,2,1,1,1,1,1,1,1,1,1,1,1,1],"backgroundColor":["#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6"],"percentage":[22.37,19.18,8.22,4.11,3.65,2.74,1.83,1.83,1.37,1.37,1.37,1.37,0.91,0.91,0.91,0.91,0.91,0.46,0.46,0.46,0.46,0.46,0.46,0.46,0.46,0.46,0.46,0.46,0.46],"barThickness":13}]},"options":{"indexAxis":"y","maintainAspectRatio":false,"scales":{"y":{"ticks":{"precision":0,"autoSkip":false,"font":{"family":"Montserrat"},"color":"#000000"}},"x":{"ticks":{"stepSize":null,"precision":0,"font":{"family":"Montserrat"},"color":"#000000"}}},"plugins":{"legend":{"position":"top","labels":{"font":{"family":"Montserrat"},"color":"#000000"}},"title":{"display":true,"text":"Publishers","font":{"size":24,"family":"Montserrat","weight":600},"color":"#000000"}}}}}
Metrics
Cite this
GOST |
RIS |
BibTex |
MLA
Cite this
GOST
Copy
Gu B. et al. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos // Nature Biotechnology. 2018. Vol. 36. No. 7. pp. 632-637.
GOST all authors (up to 50)
Copy
Gu B., Posfai E., Nybo K. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos // Nature Biotechnology. 2018. Vol. 36. No. 7. pp. 632-637.
Cite this
RIS
Copy
TY - JOUR
DO - 10.1038/nbt.4166
UR - https://doi.org/10.1038%2Fnbt.4166
TI - Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos
T2 - Nature Biotechnology
AU - Gu, Bin
AU - Posfai, Eszter
AU - Nybo, Kristie
PY - 2018
DA - 2018/06/11 00:00:00
PB - Springer Nature
SP - 632-637
IS - 7
VL - 36
SN - 1087-0156
SN - 1546-1696
ER -
Cite this
BibTex
Copy
@article{2018_Gu,
author = {Bin Gu and Eszter Posfai and Kristie Nybo},
title = {Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos},
journal = {Nature Biotechnology},
year = {2018},
volume = {36},
publisher = {Springer Nature},
month = {jun},
url = {https://doi.org/10.1038%2Fnbt.4166},
number = {7},
pages = {632--637},
doi = {10.1038/nbt.4166}
}
Cite this
MLA
Copy
Gu, Bin, et al. “Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.” Nature Biotechnology, vol. 36, no. 7, Jun. 2018, pp. 632-637. https://doi.org/10.1038%2Fnbt.4166.