Very fast CRISPR on demand
Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works..
CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.
| Errataetall: |
CommentIn: Science. 2020 Jun 12;368(6496):1180-1181. - PMID 32527814 |
|---|---|
| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2020 |
|---|---|
| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:368 |
|---|---|
| Enthalten in: |
Science (New York, N.Y.) - 368(2020), 6496 vom: 12. Juni, Seite 1265-1269 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Liu, Yang [VerfasserIn] |
|---|
| Links: |
|---|
| Anmerkungen: |
Date Completed 19.06.2020 Date Revised 14.02.2024 published: Print CommentIn: Science. 2020 Jun 12;368(6496):1180-1181. - PMID 32527814 Citation Status MEDLINE |
|---|
| doi: |
10.1126/science.aay8204 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM311070485 |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM311070485 | ||
| 003 | DE-627 | ||
| 005 | 20240214232219.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2020 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1126/science.aay8204 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1292.xml |
| 035 | |a (DE-627)NLM311070485 | ||
| 035 | |a (NLM)32527834 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Liu, Yang |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Very fast CRISPR on demand |
| 264 | 1 | |c 2020 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 19.06.2020 | ||
| 500 | |a Date Revised 14.02.2024 | ||
| 500 | |a published: Print | ||
| 500 | |a CommentIn: Science. 2020 Jun 12;368(6496):1180-1181. - PMID 32527814 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. | ||
| 520 | |a CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a H2AX protein, human |2 NLM | |
| 650 | 7 | |a Histones |2 NLM | |
| 650 | 7 | |a MRE11 protein, human |2 NLM | |
| 650 | 7 | |a CRISPR-Associated Protein 9 |2 NLM | |
| 650 | 7 | |a EC 3.1.- |2 NLM | |
| 650 | 7 | |a Cas9 endonuclease Streptococcus pyogenes |2 NLM | |
| 650 | 7 | |a EC 3.1.- |2 NLM | |
| 650 | 7 | |a MRE11 Homologue Protein |2 NLM | |
| 650 | 7 | |a EC 3.1.- |2 NLM | |
| 700 | 1 | |a Zou, Roger S |e verfasserin |4 aut | |
| 700 | 1 | |a He, Shuaixin |e verfasserin |4 aut | |
| 700 | 1 | |a Nihongaki, Yuta |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Xiaoguang |e verfasserin |4 aut | |
| 700 | 1 | |a Razavi, Shiva |e verfasserin |4 aut | |
| 700 | 1 | |a Wu, Bin |e verfasserin |4 aut | |
| 700 | 1 | |a Ha, Taekjip |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Science (New York, N.Y.) |d 1880 |g 368(2020), 6496 vom: 12. Juni, Seite 1265-1269 |w (DE-627)NLM000023450 |x 1095-9203 |7 nnns |
| 773 | 1 | 8 | |g volume:368 |g year:2020 |g number:6496 |g day:12 |g month:06 |g pages:1265-1269 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1126/science.aay8204 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 368 |j 2020 |e 6496 |b 12 |c 06 |h 1265-1269 | ||