Solution for Single-Cell CRISPR Screening Technology    

The advent of CRISPR technology has revolutionized the field of gene editing. Traditional genomics research relies on large-scale cell population data, which often fails to reveal gene functions at the single-cell level. The rise of single-cell technologies has overcome this limitation, allowing us to explore gene functions at the individual cell level. Single-cell CRISPR screening combines CRISPR technology with single-cell sequencing, enabling gene editing in single cells and evaluating the effects of these edits on cell behavior. This approach offers new perspectives for studying complex biological processes in fields such as cancer, immunology, and developmental biology.

Traditional CRISPR Screening Technologies

Traditional CRISPR screening techniques utilize high-throughput gene editing tools. By synthesizing single-guide RNAs (sgRNAs) targeting different genes and delivering them into cells using lentiviral vectors, a cell library with various gene perturbations is generated. During the screening process, cells with specific phenotypes are enriched, and the sgRNA sequences within them are amplified to identify genes associated with those phenotypes. This method is widely used in drug target screening and gene function studies.

Single-Cell CRISPR Screening Technologies

In comparison to traditional methods, single-cell CRISPR screening offers higher resolution, enabling the analysis of gene perturbation effects at the single-cell level. Here are several key single-cell CRISPR screening methods:

• Perturb-Seq: This method introduces guide barcodes (Guide Barcode, GBC) into CRISPR lentiviral vectors, obtaining sgRNA and transcriptome data for each cell through single-cell sequencing. GBC links gene perturbations with transcriptomic data, facilitating the analysis of gene regulatory networks and phenotype relationships.
• CRISP-Seq: Similar to Perturb-Seq, CRISP-Seq incorporates unique guide indices (Unique Guide Index, UGI) into CRISPR lentiviral vectors to tag sgRNAs. By combining UGI with single-cell transcriptomic data, researchers can delve into the functions and interactions of gene perturbations.
• CROP-Seq: This design inserts a U6-gRNA expression cassette into the 3' LTR region of the Puro resistance gene, simplifying sgRNA capture. This approach improves experimental efficiency by allowing direct retrieval of sgRNA information during single-cell sequencing.
• Direct Capture Perturb-Seq: This new method improves sgRNA capture by designing reverse transcription primers at both the 5' and 3' ends. It addresses issues like sequence pairing errors between the tagging sequence and sgRNA and can load multiple sgRNAs into a single lentiviral vector, greatly enhancing screening efficiency.

Figure 1. CRISPR/Cas9 combined with single-cell sequencing screening technology process

Applications of Single-Cell CRISPR Screening Technologies

• High-Throughput CRISPR Perturbations

Single-cell CRISPR screening enables high-throughput screening of extensive gene perturbations, recording and analyzing CRISPR perturbation events and their phenotypic changes at the single-cell resolution. Compared to traditional methods, this high-sensitivity and high-resolution approach allows for the identification and analysis of more gene functions and interactions, revealing complex biological mechanisms and potential new targets.

• Linking CRISPR Editing with Cell Types

Traditional CRISPR screening often overlooks differences in gene perturbation responses across cell types. Single-cell CRISPR screening combines gene editing with specific cell phenotypes, enabling researchers to identify which cell types are most sensitive to specific gene edits. This is particularly crucial in cancer research, as it can reveal how different tumor cell types respond differently to the same gene perturbations, aiding in the discovery of cancer-specific targets.

• Target Identification and Optimization for Cell Therapy (CAR-T)

In cell therapy, especially CAR-T cell therapy, target identification and optimization are critical. Single-cell CRISPR screening can help identify potential targets and optimize CAR-T cell effectiveness. By performing CRISPR interference or knockout at the single-cell level, researchers can systematically screen and evaluate different targets, improving target specificity and reducing toxicity to normal cells. For example, single-cell CRISPR screening can assess the effects of various cancer cell surface markers on CAR-T cells, optimizing CAR-T cell design and enhancing therapeutic outcomes.

• Connecting GWAS and QTL Identified Target Genes

Genome-wide association studies (GWAS) and quantitative trait locus (QTL) studies identify genetic variants associated with complex traits or diseases. Single-cell CRISPR screening can integrate these findings with functional genomics by validating candidate gene functions identified in GWAS and QTL studies at the single-cell level. This approach helps confirm findings from GWAS and QTL studies and provides deeper insights into the mechanisms of these genes in specific biological contexts.

• Drug Target Screening and Drug Resistance Mechanisms

Single-cell CRISPR screening is valuable in drug target screening. By performing CRISPR interference on drug targets, it allows for the assessment of drug effects on different cell types and reveals mechanisms of drug resistance. This method can identify new targets early in drug development and evaluate drug responses to different gene perturbations, improving drug design and optimizing treatment strategies.

Single-Cell CRISPR Screening Technical Challenges

Single-cell sequencing generates large and complex datasets, requiring advanced data analysis tools and algorithms to process and interpret meaningful biological information. The complexity of single-cell experiments can lead to issues with reproducibility and stability. Ensuring that each cell receives consistent CRISPR editing and obtaining accurate and reliable sequencing results are key challenges in the implementation of the technology. Additionally, single-cell CRISPR screening involves high technical costs, including sample preparation, sequencing, and data analysis. Despite decreasing costs over time, the high expenses still limit its widespread use in some laboratories and clinical settings.

Creative Biogene's Single-Cell CRISPR Screening Solutions

Creative Biogene is committed to addressing the challenges of single-cell CRISPR screening technology with effective solutions. We provide advanced computational platforms for data cleaning, integration, and bioinformatics analysis to extract meaningful biological insights. Our rigorous quality control enhances experimental reproducibility and stability, while technological innovations and process improvements lower costs. Additionally, we offer tailored technical consulting, optimal solution design, and technical training to ensure reliable and efficient research processes.

Choosing Creative Biogene means obtaining comprehensive services to enhance research efficiency and advance gene function research and disease mechanism exploration.