After a selection pressure is applied, genomic DNA is extracted from surviving or enriched cells. PCR amplifies the integrated sgRNA sequence using primers flanking the guide. This adds sequencing adapters and sample indexes. The resulting library is sequenced, and guide counts are compared between baseline and endpoint samples to calculate depletion or enrichment scores for each gene.
NGS - Next Generation Sequencing
CRISPR Screen Library and Downstream NGS Preparation
CRISPR screen library reagents and downstream NGS preparation kits for reliable guide readout
Reliable NGS preparation is what protects the fidelity of your pooled CRISPR screen. When you run a pooled CRISPR screen, you need to quantify guide representation by sequencing. The NGS preparation step is where screen fidelity is won or lost.
CRISPR screen library reagents and downstream sequencing preparation kits designed to match them support the full pooled screen workflow you run, from guide library to guide-count readout. NGS prep kits for sgRNA libraries are optimised for common lentiviral guide expression vector backbones. They support multiplexed readout across your batches of samples, with supplementary primer sets available for expanded indexing.
Custom sgRNA library cloning is available for your non-standard guide designs. Sequencing services accept DNA extracted from your post-screen cells and return raw sequencing data at standard or high read-depth tiers. All sequencing preparation kits are compatible with Illumina short-read instruments.
- Library vector backbone. NGS prep kits are matched to specific lentiviral guide expression vector backbones, and the kit that matches the backbone used in your screen ensures primer compatibility.
- Number of samples. For up to 48 samples per run, a standard NGS prep kit covers your needs. For more than 48 samples, or for non-standard indexing, a supplementary primer set gives you expanded multiplexing.
- Sequencing in-house vs. service. For in-house sequencing, the NGS prep kit runs on your own instrument. For send-away sequencing, the NGS sequencing service gives you standard or high read-depth tiers, and it works with compatible sgRNA library backbones.
- Custom guide design. If your guides are not covered by a standard library, the custom sgRNA library cloning service builds an end-to-end library for non-standard or specialised guide designs. Pricing is project-dependent. Contact BioCat for a quote.
Applications
Genome-wide loss-of-function screens
Pooled Cas9 knockout libraries score guide fitness across cancer lines, T cells, and organoids, paired with CRISPR-Cas9 reagents.
CRISPRa and CRISPRi screens
Activation and interference libraries use dCas9 fusions to modulate expression without cutting DNA, revealing regulators knockout screens miss.
Drug target and resistance screens
Screens run under small-molecule pressure map genetic drivers of drug sensitivity and resistance, informing combination therapy design.
Immune cell functional genomics
Screens in T cells, NK cells, and macrophages identify regulators of cytotoxicity and cytokine output, relevant to immunotherapy research.
High-read-depth sequencing service
For rare guides or low fold-change hits, a deeper tier adds read depth so you do not need extra in-house capacity, via sequencing services.
Custom guide library screens
When your target genes sit outside a standard library, custom sgRNA cloning builds an end-to-end library, paired with gene synthesis.
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DNA-seq from Genetic Screens
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Frequently asked questions
A commonly applied rule is 300 to 500 reads per guide at baseline. This gives reliable quantification of guide abundance. For a genome-wide library of 80,000 guides at 500x coverage, you need roughly 40 million reads per sample. Deeper sequencing improves sensitivity for guides targeting essential genes with modest effects.
False positives arise from PCR amplification bias, low library representation during lentiviral transduction, off-target guide activity, and incomplete dropout of essential genes in slow-growing cells. Using two or more independent guides per gene, and statistical models that account for guide-level variance, reduces false discovery rates.
Yes. Primary cells, including T cells, macrophages, and haematopoietic stem cells, are compatible with pooled CRISPR screens. However, transduction efficiency and cell viability during selection need optimisation. Libraries may need to be smaller, or guide coverage increased, to maintain representation given lower transduction efficiencies in primary cells.
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