دورية أكاديمية
The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells
| العنوان: | The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells |
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| المؤلفون: | Mohr, Marina, Damas, Nkerorema, Gudmand-Høyer, Johanne, Zeeberg, Katrine, Jedrzejczyk, Dominika, Vlassis, Arsenios, Morera-Gómez, Martí, Pereira-Schoning, Sara, Puš, Urška, Oliver-Almirall, Anna, Lyholm Jensen, Tanja, Baumgartner, Roland, Tate Weinert, Brian, Gill, Ryan T., Warnecke, Tanya |
| المصدر: | Mohr , M , Damas , N , Gudmand-Høyer , J , Zeeberg , K , Jedrzejczyk , D , Vlassis , A , Morera-Gómez , M , Pereira-Schoning , S , Puš , U , Oliver-Almirall , A , Lyholm Jensen , T , Baumgartner , R , Tate Weinert , B , Gill , R T & Warnecke , T 2023 , ' The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells ' , ACS Synthetic Biology , vol. 12 , no. 2 , pp. 375-389 . https://doi.org/10.1021/acssynbio.2c00179Test |
| سنة النشر: | 2023 |
| المجموعة: | Technical University of Denmark: DTU Orbit / Danmarks Tekniske Universitet |
| مصطلحات موضوعية: | CRISPR, MAD7, NHEJ, HDR, Frameshift mutations, CAR T-cells |
| الوصف: | CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells. |
| نوع الوثيقة: | article in journal/newspaper |
| وصف الملف: | application/pdf |
| اللغة: | English |
| العلاقة: | https://orbit.dtu.dk/en/publications/4da7de01-4a9e-47c4-9a1b-b91f6592ce6cTest |
| DOI: | 10.1021/acssynbio.2c00179 |
| الإتاحة: | https://doi.org/10.1021/acssynbio.2c00179Test https://orbit.dtu.dk/en/publications/4da7de01-4a9e-47c4-9a1b-b91f6592ce6cTest https://backend.orbit.dtu.dk/ws/files/341113601/mohr-et-al-2023-the-crispr-cas12a-platform-for-accurate-genome-editing-gene-disruption-and-efficient-transgene.pdfTest |
| حقوق: | info:eu-repo/semantics/openAccess |
| رقم الانضمام: | edsbas.C35A69C7 |
| قاعدة البيانات: | BASE |
| DOI: | 10.1021/acssynbio.2c00179 |
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