Horizontüberhöhung
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.