A Comprehensive Guide to CRISPR Screening and Sequencing: Methods, Applications, and Challenges

Posted by kiko on March 2nd, 2023

In competition with bacteriophages, bacteria and archaea have evolved unique ways of defending, which include the CRISPR/Cas systems and the immune systems of bacteria and archaea, which resist the invasion of foreign DNA or RNA and recognize the foreign invaders and cleave them for immune defense.

CRISPR/Cas systems are now useful research tools that are easy to use and work well in the field of genetic engineering. In a CRISPR/Cas9-induced gene editing process, for example, the Cas9 enzyme specifically cleaves double-stranded DNA with the help of sgRNA (small guide RNA). And the cell achieves the target gene editing through non-homologous end joints (NHEJ) or homology-directed repair (HDR).

Meanwhile, CRISPR/Cas systems can be readily scaled up for genome-wide screening. CRISPR screening is a large-scale genetic loss-of-function screen approach that generates and screens a population of mutant cells to facilitate the discovery of key genes or genetic sequences in a particular cell type. The basic idea of CRISPR screening is to knock out every gene in the genome that could be important—and only one gene per cell.

The workflow of genome-wide CRISPR/Cas9 knockout screening and sequencing

1. sgRNA library construction: sgRNAs are computationally designed, synthesized, amplified by PCR, and cloned into a vector delivery system.

2. Screening: Introduce sgRNA, Cas9, and other necessary components to cells. Then the desired clones are selected and DNA is extracted.

3. Sequencing: PCR and next-generation sequencing

4. Measurement and Analysis: sgRNAs are recovered, analyzed, and associated genes identified

Applications of CRISPR Screening and Sequencing

To identify disease-related genes: Genome-wide CRISPR/Cas9 screening can be used to identify disease-related genes, which is important for new drug target discovery and provides strategies for treatment. For instance, CRISPR/Cas9 screening technologies are a boon for cancer therapy—target genes can be obtained and analyzed to find genes with a higher correlation of tumor cell survival ability. By suppressing the expression of these genes, the tumor cell cycle is blocked and apoptosis is induced, while normal blood cells are not affected as much. CRISPR/Cas9 screening can also be used to study metastasis-related genes, to explore how viruses invade and injure host cells, and more.

To study non-coding sequences: Non-coding DNA sequences, which compose about 98 percent of the human genome sequence, include non-coding RNA, cis- and trans-regulatory elements, introns, pseudogenes, telomeres, repetitive sequences, and so on. Studies have shown that non-coding sequences play a pivotal role in the regulation of gene expression, tumorigenesis, immune regulation, ontogenesis, and many other biological processes. Genome-wide CRISPR screening can be applied to study unknown non-coding DNA sequences that are of great significance for understanding gene regulation, disease, and biological evolution.

To study regulatory networks: CRISPR/Cas9 genome-wide screening has been widely applied in various fields of cell biology. However, the screening of phenotypes is mostly practiced within cell proliferation, viability, drug resistance, reporter gene expression, etc. More complex biological regulatory networks (such as transcriptomes and gene interactions) within cells require additional investigation. By combining CRISPR/Cas9 genomic screening technology and single-cell sequencing, the expression of sgRNA can be accurately captured, and the changes in gene transcription level in cells can be measured. In the meantime, a large amount of data can be analyzed according to the computational model, and the complex gene network can be depicted.

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