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Lenti-ONE GFP carries the transgene green fluorescent protein. Its expression can be assessed by fluorescent microscopy or by flow cytometry, thus giving valuable informations about the transduction efficiency of the vector.
Cas9 cDNA encodes for protein-9 nuclease from Streptococcus pyogenes. (Sequence, WP_010922251.1, 4128bp). Coexpression of Cas9 with an artificial single guide RNA (sgRNA) which fuses the crRNA with the tracrRNA offers the possibility to target and modify DNA sequences of interest.
In this vector, Cas9 is fused with GFP cDNA encoding a green fluorescent protein (sequence from Aequorea victoria genome, AAB02574.1, 717 pb). This fluorescent reporter protein allows monitoring expression and transduction by fluorescent microscopy or by flow cytometry.
CRISPR/Cas9 Lenti-ONE vectors allows to easily and efficiently deliver the CRISPR system (“Clustered Regularly Interspaced Short Palindromic Repeats”) into desired in vivo/in vitro cell types.
Originally discovered as part of bacterial immunity, we have now harnessed the CRISPR technology to enable specific gene editing in almost all cell lines or animals. CRISPR/Cas9 Lenti-ONE vectors allow to knock-in genes, to knock-out genes, and also to increase or decrease the expression of a target gene.
Details on the molecular mechanisms of DNA cleavage under CRISPR stewardship can further be found here (Jinek et al. 2012).
The use of lentiviral vectors for CRISPR technology remains one of the most effective and versatile methods of Cas9 and RNA complex transfer , with a broad tropism and a high flexibility of design allowing to address a wide range of challenges (Thomas, Ehrhardt, and Kay 2003).
1. Identify your desired method of transduction
GEG Tech offers multiple versatile components when looking to perform gene editing with CRISPR technology. Whether Cas9 and/or the RNA complex should be made available temporarily or permanently, our R&D team’s innovations offer multiple options and can help you determine which is right for your experiment. Furthermore, you can choose Cas9 & RNA complex with a transient or stable expression independently, that which allows for example to screen RNA complexes through a model which stably expresses Cas9.
The Cas9 protein and/or RNA complex can be also combined with a gene reporter such as GFP for easily control and tracking vector expression in the cells.
In long term expression
In stable transduction protocols, the lentiviral vector will integrate within the recipient’s genome, the machinery required for the stable expression of the Cas9 protein and/or the RNA complex.
This will guarantee that in dividing cells, they are passed along and in non-dividing cells such as neuronal cells, to be expressed at the same rate as the host’s cellular proteins. Once integrated, the production of Cas9 and/or the RNA complex can be induced for expression or consistently produced.
For more information on stable viral transduction, please see: https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools/transduction-with-lenti-one/
In transient expression
In transient transfection protocols, the lentiviral vector will NOT integrate within the recipient’s genome.
Instead, integration-deficient lentiviral vectors will lead to the generation of episomal vector genomes, which are administered to dividing cells for the RNA complex and the Cas 9 protein. This can be useful if DNA silencing or DNA editing is time dependent or to be used in a pilot study.
A second way to engineer transient transfection is through the use of Reverse Transcriptase-Deficient Lentiviral Vectors. Once the viral machinery is transduced into host cells, the RNA vector for the RNA complex is treated like native RNA, capable of being produced by host cells in either dividing cells or non-dividing cells, without the need to be integrated into the cell’s DNA.
For more information please see: https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools/transduction-with-lenti-one-trans/
2. Plan an efficient study
There are many ways to deliver the CRISPR/Cas9 and RNA complex genome machinery to cells, from physical and chemical methods like electroporation or polyfection, to more novel ways such as magnetic nanoparticles (MNPs)(Hryhorowicz et al. 2019).
When planning an experiment considering elements such as ease of use, transduction efficiency, cost and safety are important points to keep in mind that will equally define at what scale production is limited to. Finding a balance between all these factors should be motivated by aiming for larger-scale production protocols, in order to assure scientific reproducibility and accuracy.
3. Identify a cell line for “test” purposes
Whether you choose to perform In Vivo experiments such as transgenic animals or choose to establish a cell line capable of producing the components required for the cell machinery of the CRISPR/cas9 methodology, the possibility of using a primary cell line, such as the non-adhering murine erythroleukemia (MEL) cells, may actually help you in optimizing your protocol while providing you with an in vitro control (Bauer, Canver, and Orkin 2015).
Primary cells are not the only cell you can use. You can also use immortalized cell lines such as the HeLa cells or HEK293 T to optimize your production protocol.
For more information on creating a clonal cell line overexpressing a transgene, see: https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools/generate-clonal-cell-lines/
GEG Tech, a first class, innovative biotechnology company, is at the forefront of the development of cutting-edge lentiviral vectors.
Better than anyone, we understand the challenges and drawbacks which present themselves in the process of vector production. For this reason, we are dedicated to using our experience and unique methodology to offer you efficient solutions such as this Cas9-GFP vector.
We are committed to optimizing research in the genome editing field, with our extensive catalogue of high-performance lentiviral vectors. We place supreme importance on creating the highest quality lentiviral vector features, thanks to our experience and renowned technical proficiency.
With over 5,000 custom & ready-to-use lentiviral vectors and counting, GEG Tech is proud to be a key player in the advancement of genome engineering technologies.
Bibliography
Barrangou, R, and L Marraffini. 2014. “CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity.” Molecular Cell 54: 234–44.
Bauer, Daniel E, Matthew C Canver, and Stuart H Orkin. 2015. “Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9.” Journal of Visualized Experiments : JoVE, no. 95 (January): e52118–e52118.
Hryhorowicz, Magdalena, Bartosz Grześkowiak, Natalia Mazurkiewicz, Paweł Śledziński, Daniel Lipiński, and Ryszard Słomski. 2019. “Improved Delivery of CRISPR/Cas9 System Using Magnetic Nanoparticles into Porcine Fibroblast.” Molecular Biotechnology 61 (3): 173–80.
Jinek, M, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E Charpentier. 2012. “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.” Science 337 (6096): 816–21.
Thomas, Clare E, Anja Ehrhardt, and Mark A Kay. 2003. “Progress and Problems with the Use of Viral Vectors for Gene Therapy.” Nature Reviews.
