Dharmacon™ All-in-one (AIO) lentiviral libraries offer a powerful, streamlined solution for genome-wide gene knockout (CRISPRko), gene activation (CRISPRa) or gene inhibition (CRISPRi) screening.
Why choose All-in-one?
Combining the sgRNA and CRISPR effector into one vector allows for single-step delivery with only one selection step. This can save consumables, person power, and weeks of experimental time which would otherwise be spent on sequential transduction and selection steps. It also minimizes the impact on cell homeostasis and viability by reducing the amount of cell manipulation required – a particular advantage when using sensitive or difficult-to-transduce cell types.
Our Dharmacon AIO vector contains both gene-targeting sgRNA and the Cas9 effector of choice - whether for knockout (Cas9), CRISPR activation (dCas9-VPR), or CRISPR interference (dCas9-SALL1-SDS3).
Optimized for your workflow
Dharmacon AIO libraries are designed for pooled whole-genome screening, with four sgRNAs per gene as well as positive (gene targeting) and negative (non-targeting) controls in each library. The workflow is straightforward: transduce, select with puromycin, apply selective pressure (e.g. a drug treatment), and analyze sgRNA abundance via high-throughput sequencing.
In addition to the pre-designed AIO whole genome library, you can also create a custom pooled AIO CRISPR knockout or CRISPRmod (CRISPRa or CRISPRi) library using a gene list of your choice with our new Custom Pool Library Tool, providing tailored insight into your specific research question.
Figure 1: Lentiviral pooled screening workflow for all our CRISPR All-in-one libraries
This figure illustrates the steps of a pooled screen, beginning on day 1 with transduction of cells using the All-in-one CRISPR library, followed by puromycin selection for 2 to 6 days. The cells are then split into separate populations: a reference sample and an experimental sample exposed to selective pressure. After the experiment, cells from both groups are harvested for DNA extraction and library preparation, enabling NGS-based analysis of enriched or depleted sgRNAs.
Validated performance
Knockout screens using the All-in-one CRISPRko system show robust dropout of core essential genes, whereas the All-in-one CRISPRi platform resulted in reduced essential gene dropout, since gene expression is reduced but not ablated. We offer both CRISPRko and CRISPRi libraries so that you can choose the most suitable approach (gene knockout or gene knockdown) and design the most appropriate experiment to answer your biological question.
Alternatively, CRISPRko and CRISPRi screens can be used together for orthogonal validation. Our own knockout and silencing screens for vemurafenib resistance identified shared gene targets, providing stronger evidence for their biological relevance than either screen alone.
Dual-direction screening for deeper insights
CRISPRa and CRISPRi All-in-one libraries can be used for parallel activation and silencing experiments to identify gene targets that either enhance or suppress drug responses. This type of orthogonal screen could help elucidate drug responses, increase confidence in hit identification, and even identify other therapeutic treatments to enhance drug efficiency. Dual direction approaches are becoming increasingly common when analyzing drug mechanisms of action or finding new biomarkers, as well as for providing gene targets for development of combinational therapies.
From library prep to confident analysis
After performing the CRISPR screen, we offer the Dharmacon™ NGS Library Prep Kit, which is fully compatible with all our CRISPR Dharmacon™ human lentiviral pooled screening libraries and optimized for use with the Illumina® sequencing platform. This kit enables precise amplification and sequencing of sgRNA constructs from genomic DNA while minimizing amplification bias, ensuring accurate quantification of sgRNA representation. With the NGS Library Prep Kit, you can confidently attribute differences in sgRNA representation after sequencing to enrichment or depletion events from the primary screen.
Want to learn more?
Download our latest application note: All-in-one pooled CRISPR screening to accelerate drug discovery, development, and pathway analysis.