Synthetic CRISPR sgRNA libraries enable scalable genome-wide activation screening.

CRISPR-based functional genomics has transformed gene function research, enabling the targeted perturbation or modulation of hundreds to thousands of genes in a single experiment. Using CRISPR knockout (CRISPRko), activation (CRISPRa), inhibition (CRISPRi), and epigenetic silencing (CRISPRoff), researchers can map genetic pathways, uncover cellular processes, identify novel therapeutic targets, and genetically profile potential therapeutics.

When planning a large-scale CRISPR screen, the first choice is between pooled and arrayed formats.

• Pooled CRISPR screens deliver a high-throughput method to study cell-autonomous phenotypes such as cell survival, gene expression, and drug resistance, where the genetic perturbation only affects the altered cell. By introducing diverse single-guide RNAs (sgRNAs) individually via lentiviral particles to create a mixed population of cells, these screens allow the exploration of a broad set of discretely defined phenotypes. This approach is particularly useful for large-scale genetic screens and selection-based experiments; however, it requires cell selection and can require significant bioinformatics to deconvolute the data.
• Arrayed CRISPR screens offer a complementary approach in targeting genes one-by-one in individual wells. This method is simpler in design and provides a powerful platform for studying complex, non-autonomous cell phenotypes (where a genetic perturbation can affect neighboring cells) —including cell-cell interactions and secreted factors. Arrayed screens are ideal for high-content imaging and phenotypic assays that require spatial resolution, allowing researchers to create rich phenotypic profiles and observe subtle changes in cellular behavior and morphology.

The challenges of arrayed CRISPR screening

While arrayed screening offers insights into cellular behaviors and a simplicity in design that pooled screens may not allow, they have historically presented technical challenges. The construction of traditional plasmid-based arrayed libraries typically involves extensive cloning and viral packaging, which are both time- and labor-intensive.

Additional challenges include the risk of well-to-well contamination over time, variability in viral titers across wells, and the specialized biosafety requirements associated with handling lentiviruses. These technical issues have the potential to delay experimental timelines and complicate large-scale studies.

Recognizing these challenges, Yin et al. recently developed a novel, plasmid-based cloning methodology to streamline the construction of arrayed CRISPR libraries.¹ Their approach resulted in the generation of large-scale plasmid libraries for gene ablation (CRISPRko), gene activation (CRISPRa), and epigenetic silencing (CRISPRoff). Each plasmid encoded an array of four sgRNAs targeting different regions of the same gene or respective transcription start site (TSS)—a design that accommodates most human DNA polymorphisms and enhances the likelihood of successful gene perturbation.

Their strategy led to impressive outcomes, including:

• 75–99% gene ablation efficiency (CRISPRko)
• 76–92% gene silencing efficiency (CRISPRoff)
• Significant gene activation across diverse targets (CRISPRa).

The power of this approach was further demonstrated by the consistent outperformance of their quadruple-guide RNA (qgRNA) vectors compared to single-guide constructs, particularly in gene activation. To showcase the practical implications of their methodology, the team conducted a large-scale activation screen of 1,634 human transcription factors.

This led to the discovery of 11 novel regulators of the cellular prion protein (PrPC), a key player in neurodegenerative diseases. In a separate CRISPRko screen, they also uncovered five novel autophagy modifiers that were missed by other widely used libraries.

These discoveries not only validate Yin et al.’s approach, but they also highlight the potential of improved CRISPR libraries to uncover insights that might be overlooked using customary methods.

Simplifying arrayed CRISPR screens with synthetic solutions

While the advancements by Yin et al. represent a significant step forward in arrayed CRISPR technology, Revvity’s synthetic CRISPR reagents offer an even more streamlined path to successful arrayed screening. By offering pre-designed sgRNAs—available as arrays of single guides or pools of four guides targeting the same gene—these solutions eliminate the need for extensive cloning, purification, and lentiviral packaging.

Key benefits of Revvity's synthetic CRISPR reagents include:

1. Elimination of the entire plasmid workflow: These solutions remove the need for extensive cloning, purification, and lentiviral packaging. There is also no need to optimize promoter sequences or validate sequential expression of multiple guides.
2. Increased experimental flexibility: Reagents can be provided as arrayed single guides or pools of guides to each gene target, allowing researchers to tailor their approach to specific experimental needs.
3. Enhanced gene activation: Pooling CRISPRa synthetic sgRNAs to a single gene can lead to stronger gene activation, potentially increasing the sensitivity and reliability of hit identification in large-scale genetic screens.²
4. Reduced biosafety concerns: The chemically synthetic nature of these guide RNAs eliminates the need for viral packaging, which simplifies the process and reduces associated risks.

With the recent release of synthetic CRISPRa sgRNA-based whole-genome, gene-family, and custom collections, replacing previous crRNA offerings, Revvity provides a pre-designed solution for carrying out multiple modes of arrayed screening, which significantly reduces the time and effort required to initiate experiments.

Together, these synthetic CRISPR solutions represent the next evolution in arrayed screening technology, offering researchers a powerful tool to accelerate their genomic studies.

Learn more about Revvity’s Dharmacon™ CRISPRa synthetic sgRNA libraries or complete our contact form and one of our dedicated team will be in touch.