Description
General description
CRISPRi is a potent tool for modulating gene expression. This technology uses dCas9 to act as a synthetic transcription factors by recruiting endogenous transcription repressor complexes to gene promoters and enhancers, resulting in down regulated gene transcription. CRISPRi achieves LOF phenotypes without the limitations of RNAi and CRISPR KO, although CRISPRi relies on delivery by lentivirus, this technology offers new possibilities for genome engineering. CRISPRi can be used individually to target areas of the genome that are inaccessible by other gene perturbation technologies (e.g., non-coding regions) or in conjunction to uncover gene-regulatory networks underlying discrete phenotypes. Finally, pooled CRISPRi screening can be paired with single-cell RNA-sequencing methods to enable high-dimensional characterization of CRISPR gene perturbation.
Human CRISPRi Mitochondria Trafficking Motility Kitcontains 2 sub-pools of top 5 gRNAs per gene as well as a separate sub-pool of 5 supplemental gRNAs per gene for increased sensitivity and includes non-targeting controls built in. All pools are functionally titered based on # of guides to provide approximately 3 replicates depending on cell line at 500 gRNAs per cell. Vectors contain both BFP and puromycin as selection markers and an optimized scaffold for sgRNA expression and function. Each sub pool of the library includes non-targeting sgRNA controls. The UCOE KRAB-dCas9 effector is designed for consistent expression and inhibition allowing gene silencing loss-of-function screening with less risk of toxicity to your cells due to double stranded breaks which make the CRISPRi system the ideal choice for perturbation of non-coding genes, applications that call for a reversible, titratable solution, and essential genes.
Individual CRISPRi clones are easily ordered online or by contacting your local sales representative
Custom pools for follow up screening or 10x Genomics Compatible CRISPR pools are also available by contacting your local sales representative
For more information about screening with CRISPRi/a please Click Here.
Application
Functional Genomics/Target Validation
Focused forward genetic screening
Set up and optimization of screen assay
Creation of cell lines stably expressing KRAB-dCas9
Validated positive and negative controls
Biochem/physiol Actions
The power of CRISPR for genome engineering, coupled with the ability to perform large-scale, whole genome, loss-of-function (LOF) screening, has allowed for new breakthroughs identifying gene pathways in drug resistance and disease. CRISPR is most commonly used to create double-stranded breaks that often result in loss of gene function (CRISPR-KO). However, the full extent of CRISPR′s utility extends beyond just targeted cutting of DNA. Nuclease-independent applications of CRISPR provide equal targeting specificity but instead of cutting, CRISPRi allows for targeted interference of gene function by delivering transcriptional repressor domains to a specific target sequence using modified dCas9 + gRNA complexes. Gene knockdown is complementary to CRISPR-KO and CRISPRa (activation) and has distinct advantages over existing loss-of-function strategies like RNAi. We partnered with University of California San Francisco to provide the best-in-class CRISPRi screening tools available.
Features and Benefits
Complete ready to use screening kit, including targeted screening pool, effector and positive and negative control
Best in Class UCSF gRNA selection algorithm and optimized (F+E) gRNA scaffold
Ease of optimization: Validated positive control targeting RAB1A and non-targeting control for assay set up
UCOE KRAB-dCas9 for consistent effector expression across a wide variety of cell lines
Libraries provided at high functional titer, based on FACs or CFU analysis
Use puromycin and or BFP as selection markers
Gene targets from UCSF organized into functional category of Mitochondria, Trafficking, Motility
Other Notes
2 Subpools with a minimum concentration of 1x108 TU/mL(via FACS assay)
CRISPRIL7: CRISPRi Pool H4 Top5 Mitochondria Trafficking Motility
CRISPRIL8: CRISPRi Pool H4 Supp5 Mitochondria Trafficking Motility
2 Controls and 1 Effector Construct with a minimum concentration of 5x105 TU/mL(via FACS or CFU assay) CRISPRIE: KRAB-dCAS9 CRISPRi Construct Lentiviral Transduction Particles
CRISPRI01: CRISPRi Rab1a Control Lentiviral Transduction Particles
CRISPRI06: CRISPRi Nontargeting Control Puromycin Transduction Particles
Legal Information
CRISPR Use License Agreement
Lentiviral, WPRE and Evrogen Fluorophore Licenses
