Mammalian Gene Editing by CRISPR
CRISPR-Cas (Clustered regularly-interspaced short palindromic repeats) has evolved to be the prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages, and provides a form of acquired immunity. The CRISPR-Cas9 system has been adapted in mammalian system for targeted gene knock-out (KO) and Knock-in (KI).
The CRISPR system is "cheap, quick and easy to use" (Nature, 2015, Vol 522, pages 20-24) for editing gene features in mammalian cells, Lifeome configured CRISPR-Cas9 systems to make it even easier to adopt in your lab by offering:
- All-in-one Plasmid with Cas9 readily expressed, just clone your gDNA
- Ready to use, gene specific pre-designed Duo-gRNA sets, just transfect target cells
- Pre-designed gene specific Duo-gRNA sets for in vitro gRNA production
- AAV and Lentiviral CRISPR systems available
- Mouse and Rat knock-out and knock-in services, constitutive or inducible
All-in-one Cas9-gRNA Targeting System
Design and clone gDNA under U6 promotor for gRNA transcription, the lentiviral compatible All-in-one plasmid expresses Cas-9 to facilitate the CRISPR targeting. EGFP is expressed from the same vector visualize transfection efficiency.
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Materials Provided: |
Vector: pLenti-EF1a-Cas9-EGFP-U6-sgRNA |
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Convenience Factors |
- One vector to express Cas9 and gRNA
- U6 promoter driving efficient gRNA transcription
- EF1α promoter for high level Cas9 expression
- Lentiviral compatible
- EGFP for positive transfection identification |
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Vector Map |
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Brief Protocol |
- Design your gene specific gDNA sequences
- Clone gDNA sequences into pLenti-EF1a-Cas9-EGFP-U6-sgRNA
- Transident transfection into cells for gene targeting
- Visualize EGFP for transfection efficiency
- Determine gene targeting success by your methods of choice |
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Ready? |
Purchase CRISPR-Cas9 All-in-one Kit |
Gene Specific gRNA Duo Sets
For most human, mouse and rat genes, Lifeome has selected two gRNA targeting sequences and cloned into pGL3-U6 gRNA transcription vector. The sequences are selected for high effiency targeting using CRISPR-Cas9 system but low "off-target" potentials. The Duo Sets can be co-transfected with Cas9 expression plasmids into cells for effective gene targeting.
Gene Specific gRNA Duo Sets for Knock-In
For most human, mouse and rat genes, Lifeome has selected two gRNA targeting sequences and cloned into pGL3-U6 gRNA transcription vector. The sequences are selected for high effiency targeting using CRISPR-Cas9 system but low "off-target" potentials. The Duo Sets can be co-transfected with Cas9 expression plasmids into cells for effective gene targeting.
Target gene knock-in (KI) is achieved by through homologous recombination. A donor plasmid containing both left and right arms of a homologous sequences to the Cas9 nicking site, flanking a GFP-Puromycin cassette that can be replaced with a gene of interest. After the plasmids for Cas9 expression, gRNA expression and the donor plasmid are delivered into the cells, the following three steps will happen sequentially to set the GFP-Puromycin cassette into the target genomic location:
1. Cas9-gRNA complex nicks the intended location;
2. Homologous recombination mechanism will "patch" the nick with the GFP-Puromycin cassette
Cas9 Expression Clones
Both pST1374-NLS-Cas9 and pST1374-NLS-Cas9-EGFP offer consititutively high level Cas9 expression under CMV promotor, where EGFP can be used to monitor transfection efficiency. The N-terminal NLS (nucleus localization signal) will effectively direct the Cas9 expression in the nucleus where it will bind to gRNA for gene targeting.
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Cas9 Expression Vectors: |
pST1374-NLS-Cas9
OR pST1374-NLS-Cas9-EGFP |
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Materials Needed: |
Your gene specific gRNA expression vectors |
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Methods |
Co-transfection with your gene specific gRNA expression clone(s), such as Lifeome gene specific Duo Sets. |
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Ready? |
Purchase pST1374-NLS-Cas9 OR
Purchase pST1374-NLS-Cas9-EGFP |
AAV and Lentiviral Options
Lentiviral packages are available as custom services for effective Cas9-gRNA delivery and expression, either by stable integration (lentiviral) or transiently (AAV).
Please inquire on these packages and one of our specialists will discuss details with you.
Not Sure What To Do?
Although CRISPR is effient and easy to use for gene targeting, it is a multi-step procol involving gRNA design, specificity search, cloning, transfection, not to mention the viral options. It can be overwhelming to get started.
Worry no more, one of our specialist will listen to your needs, and suggest the best routs achieving your goals. Please contact our specialists HERE.
