Studying the function of genes in metabolic pathways or in the development of certain diseases is of great importance for disease mechanism research and drug discovery. CRISPR, the newest generation of gene editing technology, has become a powerful tool for conducting forward genetic screening experiments with its unique advantages of both ease of operation and versatility. CRISPR technology is used to build up a library of mutants that may be associated with certain types of functions, and to identify genes associated with such functions through functional screening and enrichment, PCR amplification and deep sequencing analysis.
CRISPR/Cas9 Library Screening Fundamentals
High-throughput screening can be divided by two types: array and hybrid. In array libraries, single or several sgRNAs are arranged in a microarray and the gene editing sequence in each well is known. However, with hybrid libraries, the sgRNA library to be screened is designed by a computer and enhanced with positive clones before being transferred to host cells to introduce various gene mutations, and then results are obtained via high-throughput sequencing or other methods. Hybrid library screening is simple, affordable, and can be used for in vivo studies. Furthermore, hybrid libraries enable comprehensive genome-wide coverage with results that can be detected after long-term culture of the infected cells. High-throughput screening of CRISPR libraries is mostly performed using the hybrid library format.
CRISPR/Cas9 Library Screening Process
- Design & Synthesizis of sgRNA Libraries
The first step in the CRISPR/Cas9 Library Screening Process is to create a library of mutations in the target gene or genes. For high-throughput screening, the size of the library is one of the most influential factors that impacts screening results. Genome-wide libraries usually contain more than 100,000 sgRNAs.
- Construction of Lentiviral Vectors and Infection of Host Cells
The most critical step in CRISPR/Cas9 high-throughput library screening is the construction of lentiviral vectors. The sgRNA library is lentivirally-packaged and transduced to Cas9-expressing cell lines at a lower multiplicity of infection, ensuring that only one virus enters each cell; thus, enabling functional screening of genes corresponding to different sgRNAs.
- Positive or Negative Screening
Another important step in CRISPR/Cas9 library screening is the selection of an appropriate screening strategy. Depending on the purpose of the study, CRISPR high-throughput screenings are divided into positive and negative screening groups.
- High-Throughput Sequencing & Bioinformatics Analysis
Bioinformatics is an essential tool for finding genes that could be candidates in the CRISPR high-throughput screening study. This technique helps researchers exclude false positives and negatives, which ensures accurate analysis results of their studies’ progress so they can make better decisions on what next steps should take place based off tested information.
Genomic DNA is extracted from selected cell subpopulations and the DNA template should be sufficient to guarantee library abundance. After extraction of genomic DNA, PCR amplification of sgRNA targeting regions is performed. These regions are then subsequently subjected to high-throughput sequencing to quantify their relative abundance. After high-throughput sequencing, the relationship between genotype and phenotype can be determined by judging whether sgRNAs are enriched or depleted based on the change in the relative abundance of sgRNAs before and after screening. Using bioinformatics tools, it is possible to determine whether a gene is significantly enriched in the same environment by assessing the level of enrichment of multiple sgRNAs in the same gene.
- Validation of Candidate Genes
After screening several candidate genes with CRISPR/Cas9 technology, a series of methods are needed to validate them, identifying the functional genes that regulate a specific phenotype. The first step is to analyze off-target effects, which may lead to false positive results if the non-target site is within an exon. Further validation of the gene is necessary to perform a single gene knockout (using CRISPR/CAS9), construct a candidate gene knockout cell line (by screening monoclonal cells), confirm that the gene has been knocked-out (after genotyping and immunostaining), and test the effect of candidate gene knockout on viral replication. Genetic complementation testing can also be performed to determine whether the effects are due to gene knockout. Genetic complementation tests can also determine the if the effect is due to the gene knockout.
CRISPR/Cas9 Library Advantages
- Wide range of applications, not limited by gene sequence, cell, or species
- Quick and accurate identification of genes and gene clusters associated with a certain phenotype
- Identification ofnew targets with genome-wide screening
- Gene modifications with low false positive and high target hit rates
CRISPR Libraries/sgRNA Libraries | Synbio Technologies
With more than ten years of experience in CRISPR library design and synthesis, Synbio Technologies can provide customers around the globe with precise customized solutions. We offer sgRNA design, oligo pool synthesis, CRISPR library/sgRNA library construction, NGS validation, and high-content screening to support your next research project. Our team enjoys solving problems. When you want to explore more, we will be ready to help!