Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice
CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as conca...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article (Journal) Chapter/Article |
| Language: | English |
| Published: |
September 30, 2021
|
| In: |
bioRxiv beta
Year: 2021, Pages: 1-37 |
| DOI: | 10.1101/2021.09.30.462539 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1101/2021.09.30.462539 Verlag, lizenzpflichtig, Volltext: https://www.biorxiv.org/content/10.1101/2021.09.30.462539v1 
|
| Author Notes: | Rebekka Medert, Thomas Thumberger, Tinatini Tavhelidse, Tobias Hub, Tanja Kellner, Yoko Oguchi, Sascha Dlugosz, Frank Zimmermann, Joachim Wittbrodt, Marc Freichel |
MARC
| LEADER | 00000caa a2200000 c 4500 | ||
|---|---|---|---|
| 001 | 1881632822 | ||
| 003 | DE-627 | ||
| 005 | 20240307091454.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 240226s2021 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1101/2021.09.30.462539 |2 doi | |
| 035 | |a (DE-627)1881632822 | ||
| 035 | |a (DE-599)KXP1881632822 | ||
| 035 | |a (OCoLC)1425217359 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rda | ||
| 041 | |a eng | ||
| 084 | |a 32 |2 sdnb | ||
| 100 | 1 | |a Medert, Rebekka |e VerfasserIn |0 (DE-588)1185586539 |0 (DE-627)1664986626 |4 aut | |
| 245 | 1 | 0 | |a Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice |c Rebekka Medert, Thomas Thumberger, Tinatini Tavhelidse, Tobias Hub, Tanja Kellner, Yoko Oguchi, Sascha Dlugosz, Frank Zimmermann, Joachim Wittbrodt, Marc Freichel |
| 264 | 1 | |c September 30, 2021 | |
| 300 | |b Illustrationen | ||
| 300 | |a 37 | ||
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a Computermedien |b c |2 rdamedia | ||
| 338 | |a Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Gesehen am 26.02.2024 | ||
| 520 | |a CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5’biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5’biotinylation. Thus, we suggest that a combination of a 5’biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome. | ||
| 700 | 1 | |a Thumberger, Thomas |e VerfasserIn |0 (DE-588)1026677068 |0 (DE-627)727045091 |0 (DE-576)354676075 |4 aut | |
| 700 | 1 | |a Tavhelidse-Suck, Tinatini |e VerfasserIn |0 (DE-588)1124483764 |0 (DE-627)878199837 |0 (DE-576)482786531 |4 aut | |
| 700 | 1 | |a Hub, Tobias |e VerfasserIn |0 (DE-588)1321752156 |0 (DE-627)1881632245 |4 aut | |
| 700 | 1 | |a Kellner, Tanja |e VerfasserIn |0 (DE-588)1124863559 |0 (DE-627)879196548 |0 (DE-576)483202142 |4 aut | |
| 700 | 1 | |a Oguchi, Yoko |e VerfasserIn |0 (DE-588)1193597021 |0 (DE-627)1672326990 |4 aut | |
| 700 | 1 | |a Dlugosz, Sascha |e VerfasserIn |0 (DE-588)1219223301 |0 (DE-627)1735161551 |4 aut | |
| 700 | 1 | |a Zimmermann, Frank |e VerfasserIn |0 (DE-588)1034438654 |0 (DE-627)745596975 |0 (DE-576)382055268 |4 aut | |
| 700 | 1 | |a Wittbrodt, Joachim |e VerfasserIn |0 (DE-588)1038250919 |0 (DE-627)756835356 |0 (DE-576)167753401 |4 aut | |
| 700 | 1 | |a Freichel, Marc |d 1968- |e VerfasserIn |0 (DE-588)17286819X |0 (DE-627)697797147 |0 (DE-576)133723615 |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t bioRxiv beta |d Cold Spring Harbor : Cold Spring Harbor Laboratory, NY, 2013 |g (2021) vom: Sept., Artikel-ID 2021.09.30.462539, Seite 1-37 |h Online-Ressource |w (DE-627)783307497 |w (DE-600)2766415-6 |w (DE-576)404299903 |7 nnas |a Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice |
| 773 | 1 | 8 | |g year:2021 |g month:09 |g elocationid:2021.09.30.462539 |g pages:1-37 |g extent:37 |a Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice |
| 856 | 4 | 0 | |u https://doi.org/10.1101/2021.09.30.462539 |x Verlag |x Resolving-System |z lizenzpflichtig |3 Volltext |
| 856 | 4 | 0 | |u https://www.biorxiv.org/content/10.1101/2021.09.30.462539v1 |x Verlag |z lizenzpflichtig |3 Volltext |
| 951 | |a AR | ||
| 992 | |a 20240226 | ||
| 993 | |a Article | ||
| 994 | |a 2021 | ||
| 998 | |g 17286819X |a Freichel, Marc |m 17286819X:Freichel, Marc |d 50000 |d 54600 |e 50000PF17286819X |e 54600PF17286819X |k 0/50000/ |k 1/50000/54600/ |p 10 |y j | ||
| 998 | |g 1038250919 |a Wittbrodt, Joachim |m 1038250919:Wittbrodt, Joachim |d 700000 |d 721000 |e 700000PW1038250919 |e 721000PW1038250919 |k 0/700000/ |k 1/700000/721000/ |p 9 | ||
| 998 | |g 1034438654 |a Zimmermann, Frank |m 1034438654:Zimmermann, Frank |d 700000 |d 710000 |e 700000PZ1034438654 |e 710000PZ1034438654 |k 0/700000/ |k 1/700000/710000/ |p 8 | ||
| 998 | |g 1219223301 |a Dlugosz, Sascha |m 1219223301:Dlugosz, Sascha |d 700000 |d 710000 |e 700000PD1219223301 |e 710000PD1219223301 |k 0/700000/ |k 1/700000/710000/ |p 7 | ||
| 998 | |g 1193597021 |a Oguchi, Yoko |m 1193597021:Oguchi, Yoko |d 140000 |e 140000PO1193597021 |k 0/140000/ |p 6 | ||
| 998 | |g 1124863559 |a Kellner, Tanja |m 1124863559:Kellner, Tanja |d 700000 |d 721000 |e 700000PK1124863559 |e 721000PK1124863559 |k 0/700000/ |k 1/700000/721000/ |p 5 | ||
| 998 | |g 1321752156 |a Hub, Tobias |m 1321752156:Hub, Tobias |d 50000 |d 54600 |e 50000PH1321752156 |e 54600PH1321752156 |k 0/50000/ |k 1/50000/54600/ |p 4 | ||
| 998 | |g 1124483764 |a Tavhelidse-Suck, Tinatini |m 1124483764:Tavhelidse-Suck, Tinatini |d 700000 |d 721000 |e 700000PT1124483764 |e 721000PT1124483764 |k 0/700000/ |k 1/700000/721000/ |p 3 | ||
| 998 | |g 1026677068 |a Thumberger, Thomas |m 1026677068:Thumberger, Thomas |d 700000 |d 721000 |e 700000PT1026677068 |e 721000PT1026677068 |k 0/700000/ |k 1/700000/721000/ |p 2 | ||
| 998 | |g 1185586539 |a Medert, Rebekka |m 1185586539:Medert, Rebekka |d 50000 |d 54600 |e 50000PM1185586539 |e 54600PM1185586539 |k 0/50000/ |k 1/50000/54600/ |p 1 |x j | ||
| 999 | |a KXP-PPN1881632822 |e 4491453403 | ||
| BIB | |a Y | ||
| JSO | |a {"origin":[{"dateIssuedDisp":"September 30, 2021","dateIssuedKey":"2021"}],"relHost":[{"origin":[{"dateIssuedKey":"2013","dateIssuedDisp":"2013-","publisherPlace":"Cold Spring Harbor","publisher":"Cold Spring Harbor Laboratory, NY"}],"type":{"bibl":"edited-book","media":"Online-Ressource"},"language":["eng"],"note":["Gesehen am 14.04.2014"],"title":[{"subtitle":"the preprint server for biology","title_sort":"bioRxiv beta","title":"bioRxiv beta"}],"physDesc":[{"extent":"Online-Ressource"}],"recId":"783307497","disp":"Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in micebioRxiv beta","titleAlt":[{"title":"bio archive"}],"pubHistory":["2013 -"],"id":{"eki":["783307497"],"zdb":["2766415-6"]},"part":{"text":"(2021) vom: Sept., Artikel-ID 2021.09.30.462539, Seite 1-37","extent":"37","pages":"1-37","year":"2021"}}],"person":[{"display":"Medert, Rebekka","family":"Medert","given":"Rebekka","role":"aut"},{"role":"aut","given":"Thomas","family":"Thumberger","display":"Thumberger, Thomas"},{"display":"Tavhelidse-Suck, Tinatini","role":"aut","given":"Tinatini","family":"Tavhelidse-Suck"},{"display":"Hub, Tobias","role":"aut","given":"Tobias","family":"Hub"},{"family":"Kellner","role":"aut","given":"Tanja","display":"Kellner, Tanja"},{"display":"Oguchi, Yoko","role":"aut","given":"Yoko","family":"Oguchi"},{"family":"Dlugosz","given":"Sascha","role":"aut","display":"Dlugosz, Sascha"},{"display":"Zimmermann, Frank","family":"Zimmermann","given":"Frank","role":"aut"},{"display":"Wittbrodt, Joachim","family":"Wittbrodt","given":"Joachim","role":"aut"},{"role":"aut","given":"Marc","family":"Freichel","display":"Freichel, Marc"}],"title":[{"title_sort":"Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice","title":"Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice"}],"language":["eng"],"type":{"bibl":"chapter","media":"Online-Ressource"},"note":["Gesehen am 26.02.2024"],"recId":"1881632822","physDesc":[{"extent":"37 S.","noteIll":"Illustrationen"}],"name":{"displayForm":["Rebekka Medert, Thomas Thumberger, Tinatini Tavhelidse, Tobias Hub, Tanja Kellner, Yoko Oguchi, Sascha Dlugosz, Frank Zimmermann, Joachim Wittbrodt, Marc Freichel"]},"id":{"doi":["10.1101/2021.09.30.462539"],"eki":["1881632822"]}} | ||
| SRT | |a MEDERTREBEEFFICIENTS3020 | ||