Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout
The recently discovered CRISPR/Cas9 system is widely used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategie...
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| Main Authors: | , , , , , , , , , , , , |
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Elsevier,
2018-12-01T00:00:00Z.
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| 042 | |a dc | ||
| 100 | 1 | 0 | |a Yvonne Knopp |e author |
| 700 | 1 | 0 | |a Franziska K. Geis |e author |
| 700 | 1 | 0 | |a Dirk Heckl |e author |
| 700 | 1 | 0 | |a Stefan Horn |e author |
| 700 | 1 | 0 | |a Thomas Neumann |e author |
| 700 | 1 | 0 | |a Johannes Kuehle |e author |
| 700 | 1 | 0 | |a Janine Meyer |e author |
| 700 | 1 | 0 | |a Boris Fehse |e author |
| 700 | 1 | 0 | |a Christopher Baum |e author |
| 700 | 1 | 0 | |a Michael Morgan |e author |
| 700 | 1 | 0 | |a Johann Meyer |e author |
| 700 | 1 | 0 | |a Axel Schambach |e author |
| 700 | 1 | 0 | |a Melanie Galla |e author |
| 245 | 0 | 0 | |a Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout |
| 260 | |b Elsevier, |c 2018-12-01T00:00:00Z. | ||
| 500 | |a 2162-2531 | ||
| 500 | |a 10.1016/j.omtn.2018.09.006 | ||
| 520 | |a The recently discovered CRISPR/Cas9 system is widely used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategies. Because current transient expression methods may still suffer from cytotoxicity and/or low efficiency, we developed non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles for targeted gene knockout. By redirecting the gammaretroviral packaging machinery, we transiently delivered Streptococcus pyogenes Cas9 (SpCas9) mRNA and single-guide RNA transcripts into various (including primary) cell types. Spatiotemporal co-delivery of CRISPR/Cas9 components resulted in efficient disruption of a surrogate reporter gene, as well as functional knockout of endogenous human genes CXCR4 and TP53. Although acting in a hit-and-run fashion, knockout efficiencies of our transient particles corresponded to 52%-80% of those obtained from constitutively active integrating vectors. Stable SpCas9 overexpression at high doses in murine NIH3T3 cells caused a substantial G0/G1 arrest accompanied by reduced cell growth and metabolic activity, which was prevented by transient SpCas9 transfer. In summary, the non-integrating retrovirus-based vector particles introduced here allow efficient and dose-controlled delivery of CRISPR/Cas9 components into target cells. Keywords: CRISPR/Cas9 all-in-one particle, transient delivery, cytotoxicity, gammaretroviral MS2 chimera, targeted gene knockout, genome editing | ||
| 546 | |a EN | ||
| 690 | |a Therapeutics. Pharmacology | ||
| 690 | |a RM1-950 | ||
| 655 | 7 | |a article |2 local | |
| 786 | 0 | |n Molecular Therapy: Nucleic Acids, Vol 13, Iss , Pp 256-274 (2018) | |
| 787 | 0 | |n http://www.sciencedirect.com/science/article/pii/S2162253118302452 | |
| 787 | 0 | |n https://doaj.org/toc/2162-2531 | |
| 856 | 4 | 1 | |u https://doaj.org/article/2595b6871a8e4673b10f1cd8093290c6 |z Connect to this object online. |