Introduction to CRISPR Screen Sequencing
CRISPR Screen technology is used for high-throughput screening, allowing a large number of gene mutant cells to be created and a mutant cell library to be screened in various external environments. The relationship between phenotype and genotype can be further evaluated using high-throughput sequencing and bioinformatics analysis. Furthermore, high-throughput screening based on the CRISPR/Cas9 library overcomes the limitations of RNA interference screening technology, such as low transfection efficiency and the capacity to repress gene expression only at the mRNA level. In the era of precision medicine, it has a lot of scientific research value and a lot of application space.
The CRISPR Screen is a high-throughput molecular target screening method. A successful CRISPR Screen begins with a well-designed sgRNA library that targets the desired genes or loci. sgRNA is designed based on sequence, then synthesized, then cloned to vector or transcribed into RNA for cell transfection. Then, using commonly used positive or negative selection, run a screen. Following the physical separation of cells into two or more populations, PCR amplification and massively parallel sequencing will be carried out. The sgRNA and targeted genes of concern will be detected after data analysis.
Types of CRISPR Screen
There are several types of CRISPR screen which includes the following
1. CRISPR KO screening with complete loss of gene expression allows for the largest phenotypic effect window and the highest statistical power for hit discovery.
2. CRISPRi screening suppresses expression rather than completely knocking out the target gene, making it ideal for studying druggability and assessing the function of genes that are essential or amplified when knocked out.
3. CRISPRa screening amplifies gene expression in its natural setting, allowing researchers to study activation-related responses on a genome-wide scale for the first time.
Workflow of Wide CRISPR/Cas9 Knockout Screening and Sequencing
1. sgRNA Library Development sgRNAs are constructed computationally, synthesized, PCR amplified, and cloned into a vector delivery system.
2. Screening Inject cells with sgRNA, Cas9, and other required components. After that, the desired clones are chosen, and DNA is extracted.
3. Sequencing PCR and next-generation sequencing
4. Analysis and measurement sgRNAs are restored, evaluated, and associated genes are discovered.
Applications of CRISPR Screening and Sequencing
Some of the applications of CRISPR screening and sequencing are the following
1. To find disease-related genes Genome-wide CRISPR/Cas9 screening can be utilized to explore disease-related genes, which is critical for the discovery of new drug targets and treatment strategies.
2. To research non-coding DNA sequences Non-coding RNA, cis- and trans-regulatory elements, introns, pseudogenes, telomeres, repetitive sequences, and other non-coding DNA sequences make up about 98 percent of the human genome sequence.
3. To investigate regulatory networks Genome-wide screening with CRISPR/Cas9 has been widely used in various fields of cell biology. However, phenotyping is mostly used in cell proliferation, viability, drug resistance, and reporter gene expression, among other things. Within cells, more complex biological regulatory networks (such as the transcriptome and gene interaction) require further investigation.
About CD Genomics
CD Genomics provides the research community with high-quality next-generation sequencing, third-generation sequencing, genotyping, microarray, and population genetics services. CD Genomics has become an influential company in the industry and continues to innovate, keeping up with the forefront of scientific research and leading the latest and most comprehensive genomics technical support.
CD Genomics has harnessed amplicon-based next-generation sequencing to create an inexpensive, reliable, and high-throughput strategy for CRISPR Screen sequencing to meet the emerging needs of research communities CRISPR Screen sequencing service can provide direct and precise data about sgRNA and targeted gene analysis and functional enrichment analysis, to assist researchers to study genes functions in a high-throughput fashion.
