A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression
Copyright © 2020 Ng et al..
We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:10 |
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| Enthalten in: |
G3 (Bethesda, Md.) - 10(2020), 6 vom: 01. Juni, Seite 1893-1901 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Ng, William A [VerfasserIn] |
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| Links: |
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| Themen: |
CRISPR/Cas9 |
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| Anmerkungen: |
Date Completed 21.06.2021 Date Revised 21.06.2021 published: Electronic Citation Status MEDLINE |
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| doi: |
10.1534/g3.120.401299 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM308973178 |
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| 100 | 1 | |a Ng, William A |e verfasserin |4 aut | |
| 245 | 1 | 2 | |a A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression |
| 264 | 1 | |c 2020 | |
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| 500 | |a Date Revised 21.06.2021 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2020 Ng et al. | ||
| 520 | |a We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight (hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype | ||
| 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 | 4 | |a CRISPR/Cas9 | |
| 650 | 4 | |a gene overexpression | |
| 650 | 4 | |a hindsight/RREB-1 | |
| 650 | 4 | |a mutant screen | |
| 700 | 1 | |a Ma, Andrew |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Molly |e verfasserin |4 aut | |
| 700 | 1 | |a Reed, Bruce H |e verfasserin |4 aut | |
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