CRISPR library is a high-throughput gene screening solution based on CRISPR-Cas9 technology. CRISPR-Cas9 system is one of the best characterized and most commonly used CRISPR-Cas systems, which consists of single-guide RNA (sgRNA) and RNA-guided Cas9 endonuclease. The sgRNA is derived from a fusion of the CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 endonuclease and the sgRNA form a complex to bind and cleave the DNA target. CRISPR-Cas9 system is a powerful tool for gene editing that can target almost any gene by designing sgRNAs with different sequences. Due to the advantages of flexible targeting, high editing efficiency, and easy operation, CRISPR-Cas9 technology has a wide number of applications in many areas including medicine, agriculture, and biotechnology.
A CRISPR-Cas9 sgRNA library typically contains thousands to tens of thousands of sgRNAs, with several sgRNAs per target gene. In a CRISPR screening experiment, the sgRNA library is introduced into target cells to create a population of mutant cells that are then screened for the desired phenotypes. sgRNA libraries can be designed to target specific genes or whole genomes without restriction by sequence, cell type, or species. This enables high-throughput functional genetic research and rapid and accurate identification of genes associated with phenotypes of interest.
When libraries are constructed, specific genes or gene families can be knocked out to understand their roles in cell growth, differentiation, metabolism, or other biological processes.
sgRNA libraries can be used for genome editing and modification to provide technical support for gene therapy and genetic disease intervention.
Large-scale gene knockout and functional screening can be achieved using sgRNA libraries to explore gene functions and interactions.
sgRNA libraries can be used to screen disease-related genes to study diseases or discover potential drug targets for new drug development.
After the target gene and the corresponding sgRNA sequence are identified, the customized oligonucleotide pools are synthesized using a high-throughput synthesis platform. Subsequently, the oligonucleotides are amplified and efficiently cloned into the appropriate lentiviral expression vector backbone. The pooled plasmid libraries can be packaged into lentiviral particles for gene screening in any selected cell line.
Amerigo Scientific offers sgRNA library construction services including oligonucleotide synthesis, amplification, and sequence cloning into plasmids. The delivered plasmids are ready for virus packaging.
Workflow for Constructing sgRNA Libraries
A library consisting of 63,950 sgRNA sequences was constructed in this case. The error rate, coverage, and uniformity of the library were detected by Sanger sequencing and NGS analysis.
30 clones were randomly selected as a batch for Sanger sequencing, and the result showed that 100% of the sgRNA sequences in the selected clones matched the theoretical sequences.
Coverage and distribution uniformity are important indicators for evaluating the quality of a library. The coverage refers to the proportion of actually detected sequences to designed target sequences contained in the library, and the distribution uniformity is used to evaluate the distribution of sequences in the library.
NGS analysis indicated 100% coverage of this sgRNA library. In addition, skew ratio less than 3 illustrated the uniform distribution of desired sgRNAs in the library, ensuring maximum screening efficiency and identification of a hit.
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