Revvity's Dharmacon All-in-one lentiviral platform has now expanded to include whole-genome library options for CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa). Like the individual All-in-one reagents, the whole-genome libraries utilize a single vector lentiviral system with all of the components necessary for CRISPRko, CRISPRa, and CRISPRi whole-genome pooled screening.
Benefits of the Dharmacon All-in-one CRISPR platform
- No need to create a stable Cas9 expressing cell line, providing significant time savings of 10 to 30 days
- Ability to target tens of thousands of genes with simple set up (no need for automation)
- Highly compatible format to work in difficult-to-transfect or primary cells that are usually not tolerant of multiple rounds of selection
Try our All-in-one CRISPR lentiviral libraries to avoid the following challenges of working with plasmid libraries
- Avoid Library Amplification: The efficiency of amplification steps can vary, leading to biases in sgRNA representation, it’s important to keep in mind PCR cycles to prevent introducing bias
- No waiting for colony growth: lose track of it and intercolony competition can result in additional bias
- No NGS to confirm representation of sgRNA sequences – and this is before you start the screening process
- No steps to avoid viral contamination: Ensuring that the viral preparations are free from contaminants, you need to use endotoxin-free plasmid purification before packaging into lentivirus
- No tedious viral packaging: Before starting you need to understand how much antibiotic to use to avoid selection bias, random integration of lentiviral constructs can lead to positional effects on gene expression, which may influence the outcomes of functional screen
- No lentiviral plasmid transfection optimization: Determine the correct cell density to maximize transduction efficacy while minimizing cell toxicity
All-in-one CRISPR lentiviral vectors for CRISPR knockout, CRISPR activation, and CRISPR interference
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The system is a lentiviral sgRNA vector including all necessary components for S. pyogenes CRISPR-Cas9 gene editing. Each sgRNA vector contains a gene-specific single guide RNA (sgRNA) and Cas9 nuclease. Additionally, the vector is available in two configurations enabling antibiotic resistance or fluorescence sorting. It is supplied packaged into lentiviral particles for immediate transduction of mammalian cells grown in culture. See the diagrams above for specifics for respective technologies: Edit-R = CRISPRko, CRISPRmod dCas9-VPR = CRISPRa, CRISPRmod dCas9-SALL1-SDS3 = CRISPRi
Ensuring confidence in screening results with tight distribution and proper representation
High representation of single guide RNAs (sgRNAs) in lentiviral pooled screening is vital for several reasons
- Guarantees diverse gene coverage, which is crucial for accurately identifying genes involved in specific biological processes
- High sgRNA representation minimizes sampling bias, leading to more reliable results and high-confidence conclusions about gene functions
- Prevents the outgrowth of dominant clones, maintaining the diversity of the sgRNA library and the validity of the findings
To have a successful screen you need to start with a high-quality library, with adequate sgRNA representation. This will help ensure the screen is unbiased and even.
Learn more about the Dharmacon All-in-one CRISPR platform on our applications page or view our related webinar available On-demand now.