CRISPR-Cas9 technology, as a “magic” weapon in a geneticist’s toolbox, can help researchers conduct convenient, fast, and accurate gene editing. CRISPR libraries are widely used in functional gene screening, drug target screening, virus target screening, revealing signal pathways, and gene interaction screening.
The main technology principles and processes of CRISPR library are as follows:
1. sgRNA Design
Efficient sgRNA design is the premise of accurate gene editing. First, it must be assured that the target sequence of 20 nucleotides is unique in the genome and will not coincide with other parts of the genome. Second, it must be ensured that the target sequence appears upstream of the PAM sequence (NGG) recognized by the Cas9 nuclease. The designed sgRNA should have low off-target effects and mediate the Cas9 nuclease efficiently. The Cas9 nuclease is composed of RuvC- and HNH-domains and cleaves target genomes to form DNA double-strand breaks (DSBs).
2. CRISPR Library Synthesis and Construction
There can be up to hundreds of thousands of sgRNAs in a CRISPR library. The development of chip-based DNA synthesis technology has greatly reduced the cost of single-base synthesis and has promoted the application of high-throughput library synthesis. To obtain an effective, efficient CRISPR-Cas9 system, oligo pools can be constructed in two-plasmid or single-plasmid systems. The two-plasmid system constructs sgRNA and the Cas9 gene in two separate plasmids, while the single-plasmid system constructs sgRNA and the Cas9 gene in the same plasmid.
3. Virus Packaging
Lentiviruses are often used in virus packaging. Lentiviral vectors provide the means to yield stable integration of genetic components for the sustained expression or knockdown of genes of interest. Due to their utility in producing stable vectors for integration into a host genome, lentiviral constructs have been used extensively in the functional analysis of genes. Lentiviral gene transfer allows for stable and conditional transgene expression in a wide variety of target cells, including non-dividing hematopoietic stem and neuronal cells. Lentiviral infection has advantages over alternative gene delivery methods including; high-efficiency infection of dividing and non-dividing cells, long-term stable expression of a transgene, and low immunogenicity.
Virus-packaged CRISPR libraries have low MOI values ( guaranteeing one virus entry per cell). According to the purpose of the experiment, the cells are processed accordingly, and finally enriched by different screening methods (forward screening or reverse screening).
5. Bioinformatics Analysis
Based on next-generation sequencing (NGS), the sgRNA sequences that play a role can be quickly identified to study the function of target gene sequences. Gene expression levels, gene mutation rates, and clinical prognosis can be analyzed by utilizing professional databases such as, PRECOG, TCGA, and COSMIC
CRISPR Library Services and Products | Synbio Technologies
Synbio Technologies stays fully engaged in advancing the research and development of CRISPR gene editing technology by continuously improving our products and services. We proudly offer many customized CRISPR library construction services, including genome-wide human, livestock and poultry knockout libraries , and microbial genome-wide CRISPR interference & activation libraries. Cutting-edge design and standardized library construction at Synbio Technologies gives you high quality products that significantly reduce research costs and drastically shorten product delivery cycles.