CRISPRCas9 Library Screening Protocol

Experiment Summary

The two main forms of libraries used for high-throughput screening are array libraries and hybrid libraries. In the former, single or several sgRNAs are arrayed in microarrays or multi-well plates, and the gene editing sequence in each well is known; in the latter, the sgRNA library to be screened is designed by computer and enriched with positive clones, which are then transferred to the host cells to introduce various mutations, and the results are finally obtained by high-throughput sequencing and other methods. In contrast, hybrid libraries are inexpensive, easy to perform and can be used for in vivo studies, providing more comprehensive coverage of the entire genome, and the results can be tested after long-term culture of the infiltrated cells. CRISPR libraries are mostly screened using hybrid libraries for high-throughput screening.

Fig. 1 CRISPR/Cas9 screening process.

Procedures for CRISPR/Cas9 Library Screening

1. Designing and synthesizing sgRNA libraries

For high-throughput screening, the size of the library is one of the key factors affecting the screening results. Genome-wide libraries typically contain more than 100,000 sgRNAs that can identify multiple genes of interest.

2. Construction of lentiviral vectors and infection of host cells

The sgRNA library is lentivirally packaged and transduced into Cas9-expressing cell lines at a low viral infection multiplicity to ensure that only 1 virus enters each cell, thus realizing the functional screening against the corresponding genes of different sgRNAs.

3. Positive or negative screening

Another important step in the work is the selection of an appropriate screening strategy. Depending on the purpose of the study, CRISPR high-throughput screening is categorized into positive and negative screening.

4. High-throughput sequencing and bioinformatics analysis

The use of bioinformatics methods to find candidate genes and exclude false-positive or false-negative screening results is essential for the later analysis of CRISPR high-throughput screening study results. Genomic DNA was extracted from selected cell subpopulations, and the DNA template should be sufficient to ensure the abundance of the library. PCR amplification of sgRNA-targeted regions was performed after genomic DNA extraction, followed by high-throughput sequencing of these regions to quantify their relative abundance. Based on the change in relative abundance of sgRNAs before and after screening, it was determined whether sgRNAs were enriched or depleted, thus determining the relationship between genotype and phenotype. Bioinformatics tools can be utilized to determine whether a gene is significantly enriched in the same context by assessing the enrichment levels of multiple sgRNAs in the same gene.

5. Validation of candidate genes

After screening several candidate genes with CRISPR/Cas9 technology, they need to be validated by a series of methods in order to identify functional genes that regulate specific phenotypes. The first step is to analyze the off-target situation, which may lead to false-positive results if the off-target site is in an exon. Further validation of the candidate genes is essential, using CRISPR/Cas9 technology for individual gene knockdowns, constructing candidate knockdown cell lines by screening monoclonal cells, and detecting the effect of the candidate knockdown on viral replication after genotyping and immunostaining to confirm that the genes have been knocked out, while genetic complementation assays can be used to determine whether the effects are caused by the knockdown.

* For research use only. Not intended for any clinical use.

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CRISPRCas9 Library Screening Protocol

Experiment Summary

Procedures for CRISPR/Cas9 Library Screening

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