A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression
Abstract 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 genehindsight(hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-functionhntalleles 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..
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Preprint| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
bioRxiv.org - (2022) vom: 30. Sept. Zur Gesamtaufnahme - year:2022 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Ng, William A. [VerfasserIn] |
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| doi: |
10.1101/788372 |
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| PPN (Katalog-ID): |
XBI000632287 |
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| 245 | 1 | 0 | |a A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression |
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| 520 | |a Abstract 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 genehindsight(hnt). From approximately 45,000 F1 progeny we recovered 8 new EMS induced loss-of-functionhntalleles 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 Biology |7 (dpeaa)DE-84 | |
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| 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 | |
| 773 | 0 | 8 | |i Enthalten in |t bioRxiv.org |g (2022) vom: 30. Sept. |
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