Unlocking the Potential of CRISPR Library Gene Editing
The emergence of CRISPR library gene editing technology has revolutionized life science research, enabling the convenient screening of functional genes. This powerful tool is invaluable for delving into genetic function and disease mechanisms as well as drug discovery projects. High throughput functional gene screening holds immense promise in modern biological studies.
Fundamentals of CRISPR Library Screening
High-throughput screening libraries provide powerful tools for gene editing and research. Array library offers precut sgRNAs on a chip or plate, while hybrid library is designed with computationally generated sequences to maximize efficiency in cost and operation time. Mixed CRISPR libraries have the advantage of being able to detect genome mutations more extensively over longer periods compared to array systems, making them ideal for studying cell infection states within the body itself.
CRISPR/Cas9 Library Screening Process
- Design & synthesis of sgRNA library
High throughput screening efficiency is heavily reliant on the size of a library; larger libraries, such as those containing more than one hundred thousand sgRNAs from an entire genome, offer greater potential for identifying exciting genes.
- Construction of lentivirus vector & infection of host cells
To achieve successful CRISPR/Cas9-based gene screening, a precise construct of lentiviral vectors is essential. The sgRNA library must be delivered through the virus in order to ensure single infection per cell – enabling the cells to express only one function and allow identification of genes associated with each expressed RNA sequence.
- Positive or negative screening
Positive screening selectively enriches target phenotype cells, while negative screening can detect the genes involved in function loss. By extending duration of the experiment, negative screening may also reveal survival-related gene markers that would otherwise be missed by positive selection techniques. Comparing changes in sgRNA abundance across different time points helps compare and identify key genetic elements associated with cell viability and physiological functions.
- High throughput sequencing and bioinformatics analysis
Modern bioinformatics techniques are essential for the analysis of CRISPR high-throughput screening results, offering researchers more reliable and accurate data. To generate dependable datasets, sufficient genomic DNA templates must be extracted from select cell subpopulations before target regions of sgRNA can be amplified through PCR and sequenced on a large scale in order to measure relative abundance. Ultimately, these measurements will help uncover how genotype influences phenotype by identifying any positive or negative selection pressure applied during testing processes.
- Validate candidate genes
Analyzing the non-target sites is essential to ensuring reliable results. To knock out a single gene, CRISPR/Cas9 technology can be employed in order to construct monoclonal cell lines with candidate gene knockout capabilities. Once confirmed via genotyping and immunostaining that the desired genes were knocked out properly, its effect on virus replication must then be evaluated – an additional genetic complementation test should also provide concrete evidence as to whether these effects are due solely to said gene’s removal or something else entirely.
CRISPR Libraries| Synbio Technologies
Synbio Technologies offers an array of CRISPR libraries to meet the varied needs of its clients. These libraries are designed to provide precise, efficient, and economical gene editing solutions for research, biopharma and other applications. Our CRISPR/Cas9 libraries offer a wide range of tools and services for customizing edits to the genome. The company’s library selection includes sgRNA design software for creating specific DNA targets, cloning vectors for inserting gRNAs into cells as well as a variety of pre-made pools of gRNAs targeting different genes or gene families. Our synthetic biology platform also provides tools for analyzing the effects of gene perturbations in real time and on a genome scale. We have developed proprietary algorithms that enable efficient editing of genomes with minimal off-target effects and improved knockdown efficiency. With Synbio Technologies’ CRISPR/Cas9 libraries, it is now possible to precisely edit genes at unprecedented speed, accuracy and cost-efficiency. Furthermore, these libraries can be used to create customized transgenic organisms with desired phenotypes such as tolerance to stress factors or increased growth rate. In addition, they can also be employed in disease modeling studies or drug discovery/development projects by manipulating multiple genes simultaneously in an effort to understand their interaction networks.
Contact us to learn more about our CRISPR Libraries services.