Decoding CRISPR Screen Sequencing: Methods, Applications and Future Prospects

CRISPR/Cas9 gene editing technology is to design a complementary sgRNA sequence of about 20bp for the target sequence, which can guide CRISPR/Cas9 system to cut the target sequence specifically, and then through the nonhomologous end joining mechanism in the cell. NHEJ) and homologous recombination, HDR) are used to repair the broken DNA, which leads to the insertion or deletion of some gene sequences and realizes the directional editing of genes. CRISPR/Cas9 gene editing has been widely used in various cell lines and animal models because of its powerful genetic screening function.

What is CRISPR Screening

Screening of CRISPR/Cas9 library is to design sgRNAs targeting multiple genes by computer to form an sgRNA library, package the sgRNA library with lentivirus, then transfer it to host cells to introduce various gene mutations, and then impose some screening conditions during cell culture, resulting in the death of some cells. Finally, sgRNAs enriched in living cells are obtained by high-throughput sequencing and other methods, and their corresponding genes are analyzed, so as to identify the candidate genes that affect the phenotype.

CRISPR system, as an immune defense mechanism of bacteria, has been transformed into a powerful gene editing tool. In CRISPR screening and sequencing, firstly, a library of sgRNAs is constructed, and each SGRNA corresponds to a specific gene sequence. The library is introduced into the cell population, and different cells carry different sgRNA, so as to realize the parallel operation of many genes, such as gene knockout, activation or inhibition. Taking CRISPR-Cas9 system as an example, Cas9 protein accurately cleaves the target DNA under the guidance of sgRNA. After specific treatment (such as drug stimulation and environmental stress), genomic DNA was extracted, and the regions corresponding to sgRNA were amplified by PCR, and then the abundance of each sgRNA was determined by high-throughput sequencing. By analyzing the changes of sgRNA abundance, we can determine which genes affect cell phenotype under specific conditions, such as survival, proliferation and differentiation.

CRISPR screening and sequencing are widely used. In the biomedical field, it can be used to find drug targets, find key genes that respond to drugs through screening, and help new drug research and development. In cancer research, genes related to tumor occurrence, development and metastasis can be identified, which provides a theoretical basis for precise treatment of cancer. In the research of basic biology, we can analyze cell signal pathways, gene regulatory networks, etc., and deepen our understanding of life processes. Because of its Qualcomm, high sensitivity and specificity, CRISPR Screening provides a powerful tool for gene function research and disease mechanism exploration, which greatly promotes the progress of life science and medicine.

Schematic of the RNA-guided Cas9 nuclease (Qi et al., 2016)

How Does CRISPR Screening Work

CRISPR screening is a method that combines the editing technology of CRISPR gene and high-throughput sequencing technology to systematically analyze gene function in the whole genome. The basic process is as follows.

Design and synthesis of sgRNA library: Determine the list of genes to be studied, and select appropriate sgRNA design software (such as CRISPR-FOCUS, CHOPCHOP, CRISPR library designer, Cas-designer, etc.). In order to ensure that genes can be edited, 4-6 sgRNA are usually designed for each gene.

Construction of lentivirus vector and infection of host cells: The sgRNA library was packaged with lentivirus and transduced into Cas9 expression cell line with low virus multiplicity of infection, ensuring that only one virus entered each cell, so that only one gene was edited in each cell.

Positive or negative screening: According to different research purposes, CRISPR Qualcomm screening can be divided into positive screening and negative screening. Positive screening refers to the application of certain screening pressure (such as drugs), and only resistant cells survive after library disturbance. The edited genes of such cells may be cell survival inhibitory genes. Negative screening is to select the surviving cells at different screening time points, obtain the missing sgRNA by comparing the abundance difference of sgRNA in cells, and analyze the corresponding genes, which may be necessary genes for cell survival.

High-throughput sequencing and biological analysis: Genomic DNA was extracted from the selected cell subsets, then the targeted regions of sgRNA were amplified by PCR, and then the relative abundance of sgRNA was counted by high-throughput sequencing. According to the changes of sgRNA abundance before and after screening, it is determined whether sgRNA is enriched or consumed, so as to determine the relationship between the gene targeted by sgRNA and phenotype (it is necessary to inhibit survival or survival).

Verification of candidate genes: After several candidate genes are screened by CRISPR/Cas9 technology, it needs to be verified by a series of methods, so as to identify functional genes that regulate specific phenotypes. First of all, it is necessary to analyze the off-target situation. If the off-target site is in the exon, it may lead to false positive results. Then using CRISPR/Cas9 technology to knock out a single gene, constructing a candidate gene knock-out cell line by screening monoclonal cells, detecting the influence of candidate gene knock-out on virus replication after genotyping and immunostaining confirm that the gene has been knocked out, and determining whether the effect is caused by gene knock-out through genetic complementation test.

General process for pooled CRISPR screens (Zhao et al., 2022)

CRISPR Screening Applications

CRISPR Screening is a powerful functional genomics technology, which is widely used in gene function research, drug target discovery, cancer mechanism exploration, cell development regulation and other fields, and provides important research means and ideas for life science and medical research.

Screening genes related to tumor occurrence and development: Through whole genome CRISPR/Cas9 screening, genes related to tumor cell survival, proliferation, metastasis and drug resistance can be found. For example, screening in hepatocellular carcinoma cell lines can identify genes related to sorafenib resistance, such as phosphoglycerate dehydrogenase (PHGDH), provide potential targets for cancer treatment, and also help to deeply understand the occurrence and development mechanism of cancer and lay the foundation for developing new treatment strategies.

To study the immune escape mechanism of cancer: Using CRISPR Screening technology, genes that regulate immune markers can be screened in tumor cells. For example, in single cell screening in vitro, it was found that CMTM6 is the stabilizer of PD-L1, and TRAF3 is the negative regulator of antigen presentation. We can also reveal the mechanism of cancer immune escape through in vitro bicellular co-culture, transplanted in vivo screening and in situ direct in vivo screening, and provide targets for immunotherapy.

Study the pathogenic mechanism of infectious diseases: Qualcomm quantity is used to identify the risk factors related to infectious diseases. By transducing sgRNA library into cells, different genes of a large number of cells are knocked out, activated or inhibited, and then the whole genome DNA of enriched cells is obtained through specific screening, which is used to amplify sgRNA gene fragments and conduct high-throughput sequencing. Finally, bioinformatics analysis is carried out on the enriched or consumed sgRNA, so as to explore the candidate genes related to phenotype, which is helpful to deeply understand the pathogenic mechanism of infectious diseases.

Discovery of pathogenic genes: the sgRNA library for the whole genome was constructed and introduced into cells or model organisms from patients for screening. We can find out the key genes leading to genetic diseases, determine their pathogenic mechanisms, and provide evidence for the diagnosis and treatment of genetic diseases. For example, through CRISPR screening technology, genes closely related to the occurrence and development of diseases have been found in some hereditary disease models.

Exploring therapeutic targets: After the pathogenic genes are identified, this technology can be further used to explore potential therapeutic targets and provide support for the development of gene therapy methods. For example, screening out genes that can correct the function of disease-causing genes or regulate disease-related pathways provides direction for designing accurate treatment strategies.

Validation and characterization of hits from noncoding CRISPR screens (Klann et al., 2018)

Advantages and Disadvantages of CRISPR Screening

CRISPR Screening is a technology combining CRISPR technology with high-throughput sequencing, which can be used to systematically study gene functions and search for disease-related genes in the whole genome. The advantages and disadvantages are as follows.

CRISPR Screening Advantages

Genome-wide coverage: It can be screened in the whole genome, not limited by the preset gene list, and it can analyze the function of a large number of genes at the same time, which is helpful to discover new genes and signal pathways.

High resolution: It can accurately locate to a single base level, accurately identify the location and type of gene editing events mediated by CRISPR, and provide detailed gene function information.

Qualcomm quantity: Combined with high-throughput sequencing technology, a large number of samples can be processed at the same time, massive data can be obtained quickly, screening efficiency can be improved, and rich experimental results can be obtained in a short time.

Good repeatability: CRISPR technology has high accuracy and repeatability. As long as the experimental conditions are properly controlled, the results of different laboratories have good consistency, which is convenient for the verification and promotion of research results.

Direct function verification: After editing the gene by CRISPR technology, the correlation between editing effect and phenotype can be directly detected by sequencing, which can directly verify the gene function and provide a powerful means for gene function research.

CRISPR/Cas9 in HSPCs and B cells (Kim et al., 2021)

CRISPR Screening Disadvantages

Off-target effect: CRISPR-Cas system may cut at non-target sites, leading to off-target editing. This may interfere with the accuracy of the experimental results and produce false positive or false negative results. It is necessary to eliminate the influence of off-target effect through bioinformatics methods and experimental verification.

Complex data analysis: The generated marine sequence data need professional bioinformatics knowledge and tools for analysis and processing. It is difficult to interpret data, and many factors need to be considered, such as gene editing efficiency, background noise, differences between samples, etc., which requires researchers' data analysis ability.

Cell type limitation: In some cell types, the editing efficiency of CRISPR-Cas system may be low, or it is difficult to achieve stable gene editing, which will affect the screening effect and operability.

Higher cost: The experiment involves CRISPR library construction, high-throughput sequencing and other technologies, and it needs to buy related reagents, instruments and equipment, and pay for sequencing, etc. The overall experiment cost is high, and there may be some economic pressure for some research institutions or projects.

Future Prospects of CRISPR Screening

CRISPR Screening has a broad development prospect in the future. Technically, the delivery system will be safer and more efficient, the off-target problem will be solved step by step, and the accuracy and efficiency will be improved. In application, in the study of disease mechanism, it can deeply analyze the molecular mysteries of diseases such as tumors; In drug research and development, accelerate target screening and efficacy evaluation; In terms of precision medical care, it helps to screen pathogenic mutations and realize personalized diagnosis and treatment. It can also be extended to cell therapy, disease modeling and other fields.

More accurate editing: constantly improve the design method of sgRNA and develop new Cas enzymes, improve the targeting specificity, reduce the off-target effect, make gene editing more accurate and reduce the impact on non-target genes.

Efficient delivery system: Develop more efficient and safer delivery vectors for CRISPR, especially for primary cells and in vivo research, so as to improve editing efficiency and expand its application scope in different cell types and organisms.

Precision medicine: used to screen and identify gene mutations related to diseases, deeply understand the pathogenesis of diseases, and provide more accurate basis for the formulation of personalized medical programs, such as screening targeted drugs for specific gene mutations of tumor patients, or evaluating the efficacy of immunotherapy drugs on different immune cell subsets.

Disease mechanism research: In-depth study on the occurrence and development mechanism of complex diseases at the cellular and individual levels, such as exploring the drug resistance mechanism of tumor cells, the mechanism of neuron death in neurodegenerative diseases, and the abnormal function of immune cells in immune system diseases.

Data analysis and processing: With the increasing amount of data generated by CRISPR Screening technology, it is necessary to develop more powerful bioinformatics tools and algorithms for data analysis, mining and interpretation, so as to extract valuable biological information, such as gene regulatory networks and cell fate determinants.

Factors impacting CRISPRi repression efficacy (Vercauteren et al., 2024)

Conclusions

As a cutting-edge gene function research technology, CRISPR Screening can accurately edit and screen the genome on a large scale with the help of CRISPR-Cas system, and combined with high-throughput sequencing technology, it can efficiently and comprehensively identify genes related to specific phenotypes. This technology has obvious advantages such as high throughput, relatively low cost and genome-wide screening. However, the off-target effect of sgRNA may lead to false positive results, and the difference between cell model and real environment in vivo may affect the accuracy of screening. Looking forward to the future, with the continuous innovation and improvement of technology, CRISPR screening and sequencing is expected to play an important role in a wider range of fields, further promote the development of life sciences and medicine, and provide more effective tools and strategies for solving human health problems.