CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing
The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), activ...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/7/3741 |
| _version_ | 1797538886859096064 |
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| author | Nina Reuven Julia Adler Nadav Myers Yosef Shaul |
| author_facet | Nina Reuven Julia Adler Nadav Myers Yosef Shaul |
| author_sort | Nina Reuven |
| collection | DOAJ |
| description | The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential <i>TAF1</i> gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR. |
| first_indexed | 2024-03-10T12:37:33Z |
| format | Article |
| id | doaj.art-3edba51b397c4e68a7f42eee4648dcbe |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T12:37:33Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3edba51b397c4e68a7f42eee4648dcbe2023-11-21T14:08:33ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-04-01227374110.3390/ijms22073741CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome EditingNina Reuven0Julia Adler1Nadav Myers2Yosef Shaul3Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, IsraelDepartment of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, IsraelDepartment of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, IsraelDepartment of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, IsraelThe clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential <i>TAF1</i> gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.https://www.mdpi.com/1422-0067/22/7/3741gene targetingCRISPR/Cas9genome editingendogenous mutagenesis in cell linesco-editingscarless selection |
| spellingShingle | Nina Reuven Julia Adler Nadav Myers Yosef Shaul CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing International Journal of Molecular Sciences gene targeting CRISPR/Cas9 genome editing endogenous mutagenesis in cell lines co-editing scarless selection |
| title | CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing |
| title_full | CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing |
| title_fullStr | CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing |
| title_full_unstemmed | CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing |
| title_short | CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing |
| title_sort | crispr co editing strategy for scarless homology directed genome editing |
| topic | gene targeting CRISPR/Cas9 genome editing endogenous mutagenesis in cell lines co-editing scarless selection |
| url | https://www.mdpi.com/1422-0067/22/7/3741 |
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