Pooled CRISPR Technologies

The in4mer Platform

The in4mer CRISPR/enAsCas12a multiplex knockout platform uses arrays of four independent guide RNAs targeting the same or different genes, delivered via an all-in-one lentiviral vector. The enAsCas12a nuclease (Kleinstiver et al., 2019) offers several advantages over Cas9: it uses shorter guide RNAs (~40 nt vs ~100 nt), has a different PAM requirement (TTTV) that expands targeting space, and enables efficient multiplex knockout from a single transcript. The in4mer system was developed by the Hart and Doench labs and published in Anvar et al. (2024).

The Inzolia Library

The genome-scale Inzolia library targets ~19,700 single genes and covers >4,900 paralog combinations (pairs, triples, and quads). It is 30% smaller than typical CRISPR/Cas9 libraries while maintaining high sensitivity in identifying core and context-dependent essential genes, effectively detecting synthetic lethal and buffering genetic interactions between paralogs (Anvar et al., 2024).

What ATG-C Provides

• Build or purchase pDNA libraries
• Amplify and validate libraries (Sanger, long-read and short-read NGS)
• Generate lentiviral libraries, including concentrating liter-size lentiviral preps for all-in-one vectors
• Conduct functional batch testing in cancer cell lines
• Perform library prep, QC, and sequencing of submitted samples

Your Responsibilities

• Cell line optimization (lentiviral titering, puromycin kill curves, polybrene testing)
• Transduction of viral library
• Maintain ≥10 cell divisions post-transduction
• Collect dry cell pellets and store at −80°C
• Perform lysis and gDNA isolation
• Submit gDNA and plate map to ATG-C

Proliferation Screen Example

For a genome-wide proliferation screen using the Inzolia library (~19,700 genes + 4,900 paralog combos), typical parameters include:

• Library size: ~25,000 constructs
• Coverage: 500–1,000× per construct
• Cells needed: 12.5–25 million transduced cells per replicate
• gDNA requirement: ~200 μg per sample (at 6.6 pg/cell)

Use our in4mer screen size calculator to estimate cell numbers, gDNA requirements, and sequencing depth for your experiment.

CRISPRi (CRISPR Interference)

A catalytically dead Cas protein (dCas9) fused to repressor domains is targeted to a promoter or enhancer, creating a steric block that transiently inhibits transcription. We use functionally validated genome-wide CRISPRi libraries with dual-guide constructs and enhanced CRISPR repressors to minimize off-target silencing and toxicity (DeWeirdt et al., 2021).

CRISPRoff

dCas9 fused to both repressor and methylase domains targets gene promoters. Genes with CpG islands can be durably repressed — epigenetic repression that persists in daughter cells. This enables heritable gene silencing without DNA cutting.

CRISPRa (CRISPR Activation)

dCas9 fused to activator domains recruits transcriptional machinery to a promoter, enabling gain-of-function screens at genome scale.

Base Editing

Nickase Cas (nCas9 or nCas12a) fused to a deaminase causes C→T, A→G, or C→G conversions without double-strand breaks. Used for targeted saturation mutagenesis, allele correction, and functional variant screens.

Perturb-seq combines CRISPR-mediated perturbations with single-cell RNA sequencing. Direct-capture technology detects which perturbation(s) occurred in each cell by capturing the sgRNA sequence. These screens are typically short (1–8 days post-transduction). The approach was pioneered by multiple groups simultaneously (Dixit et al., 2016; Adamson et al., 2016; Jaitin et al., 2016; Datlinger et al., 2017).

We have had excellent results using enhanced dCas9 repressors and dual-sgRNA vectors. The technology allows for up to genome-wide RNA-seq profiling in a single experiment.

Additional readout modalities include FACS-based screens for surface marker or reporter expression, and ORF overexpression screens for gain-of-function phenotyping. For a comprehensive review of pooled CRISPR screen design, see Bock et al. (2022).