Product Description
transEDIT CRISPR-Cas9 reagents for gene editing include a range of lentiviral vectors expressing guide RNA (gRNA) and/or Cas9 nuclease or nickase.
- Optimized gRNA designs cloned into your choice of gRNA or All-in-one (gRNA plus Cas9) lentiviral vectors
- Single or paired guide RNA CRISPR strategies for gene editing
- ssODN template for precise gene editing
- Multiple vector configurations to enable dual or triple selection for enhanced efficiency
- Constitutive Cas9 expression
- Efficient delivery: transfect or transduce
CRISPR Cas9 Target Gene Sets
transEDIT target gene sets target human, mouse, or rat genes using 3 gRNA’s that have been cloned into your lentiviral vector of choice. transOMIC technologies uses the most current design algorithms to provide gRNA designs with the highest ranking efficiency and lowest number of off target effects. A non-targeting negative control is also included.
- 3 top ranking gRNA’s cloned into your lentiviral vector of choice
- Non-targeting negative control
- Available as bacterial glycerol stocks or viral particles in convenient volumes
ssODN template plus CRISPR/Cas9 Target Gene Sets – Delete, mutate or tag your gene of interest
The CRISPR-Cas9 system and single-stranded donor oligonucleotides (ssODN) can be applied to carry out precise gene editing quickly and efficiently. This strategy allows for generating point mutations, defined sequence modifications or insertion of a small tag. Target gene sets are available targeting human, mouse or rat genes.
This is a powerful technique that can be used to examine the relevance of DNA alterations in ex vivo and in vivo systems. Using this technique, various nucleotides and amino acids have been proved to be indispensable for promoter or enhancer activity, as well as for the function or regulation of enzymes, transcription factors and signaling molecules. Mutations known or suspected to be relevant to disease can also be studied.
transEDIT CRISPR-Cas9 with ssODN template target gene sets include:
- 3 top ranking gRNA’s cloned into your lentiviral vector of choice
- Cas9 lentiviral expression vector*
- Non-targeting negative control
- ssODN template
Available as bacterial glycerol stocks or viral particles in convenient volumes
*Cas9 lentiviral vector also available with your choice of selectable marker
transEDIT gRNA and Vector Designs
Optimized gRNA designs produce efficient cleavage at the targeted site while limiting off-target effects. transOMIC technologies uses the most current design algorithms to provide transEDIT™ gRNA designs with the highest ranked efficiency and lowest number of off-targets effects (Hsu et al. 2013, Wang et al. 2013). gRNA designs are provided as single gRNA for making double stranded breaks (DSBs) as well as paired gRNA for use with Cas9D10A (nickase) strategies.
transOMIC uses the following general guidelines for gRNA design and provides additional curation to select the top ranking gRNA:
(1) High efficiency of cleavage
- Protospacer adjacent motif (PAM) – design sequences target 20 nucleotides of genomic sequence immediately upstream of the PAM sequence 5′-NGG
- GC-content – between 45% and 80%
- Nucleotide position effects – including the experimentally-determined effects of single and multiple mismatches on the targeting efficiency of off-target mismatches
- Location within the gene – target mutations closest to the start codon to ensure the best chance for loss of protein function
(2) Low off-target effects
- Minimum possible homologous sites – select design with limited number of off-target sites in the genome
- Evaluate off-target sites – penalize designs with off-target sites expected to be efficiently cleaved based on number and location of mismatches
Vector Specifications:
transEDIT gRNA expression vectors provide effective delivery to a wide range of cell types including non-dividing or post-mitotic cells. Multiple selection options for use as a single-vector (All-in-one), in combination with transEDIT Cas9 nuclease vectors or in paired gRNA/nickase experiments.
Convenience
- Efficient delivery of gRNA and Cas9 in one vector
- Single marker selection
Modular for optimization
- Control Cas9 expression in your target cell line (shown to be important for efficient knockout)
- Complementary selection strategies for efficient co-expression with Cas9 or for paired gRNA/Cas9 Nickase strategy
Cas9 Expression vectors
Cas9 Nuclease and Nickase Expression Vectors
The level of Cas9 endonuclease expression has been shown to affect the frequency of generating genome-edited clones. The efficiency of delivery and resulting expression of the targeting components vary widely between cell types. Even within a given cell population, nuclease expression levels often vary significantly. Consequently, only a small fraction of cells within culture express sufficient levels of Cas9 to facilitate double-strand break activity.
Highlights:
- Transfect or transduce target cell lines with lentiviral Cas9 expression vectors- Transduction allows for efficient generation of Cas9 stable cell lines.
- Multiple Selectable markers and Reporters – Blasticidin and/or puromycin to perform antibiotic selection selecting for Cas9 expressing cells. Co-selection can be performed with gRNA expression vectors for more efficient indel production. Fluorescent reporters (ZsGreen or tRFP) in Cas9 vectors allow for FACS sorting of high expressing Cas9 cells.
- Inducible Cas9 expression allows the creation of cell lines that limits constitutive expression of Cas9 which may reduce off target effects and may also be a necessary component when knockout of an essential gene is being considered.
- Creation of stable cell lines with Cas9 or inducible Cas9 allows for more uniform expression of Cas9 which is desirable in the context of larger multiplexed screens.
- Paired gRNA with Cas9 Nickase for gene editing – Use Cas9 D10A mutant expressing vectors in combination with 2 gRNAs to produce efficient and specific gene editing (Ran et al., 2013).
Vector Specifications:
transEDIT™ Cas9 Nuclease expression vectors incorporate selection strategies to select for high Cas9 expressing cells and variable promoters to optimize expression in target cell lines. Tet-inducible Cas9 vectors have been developed to allow for more efficient control over Cas9 expression in your target cell line.
Purchase
transEDIT™ target gene sets available for human, mouse and rat genomes. Target gene sets include three gRNA and a non-targeting control gRNA in bacterial glycerol stock or lentiviral particle formats. Additionally gRNA pools are also available for pooled screnning.
Turnaround time for bacterial glycerol stocks is 2-3 weeks.
Turnaround time for viral particles is 4-5 weeks.
Please contact on info@moldiag.in for further information and pricing
