CRISPR Screening: A Breakthrough in Genetic Research

CRISPR/Cas9
It is fascinating that an ancient defense system of prokaryotes against invading viruses now became an essential tool for molecular biologists. In 2020, Emmanuelle Charpentier and Jennifer A. Doudna won the Nobel Prize in Chemistry for the development of a method for genome editing: CRISPR/Cas9 system. After their discovery in 2011, it has been used for editing parts of the genome by removing, adding, or altering sections of the DNA sequences. In an earlier post, Alper introduced it so here we will talk about its use in genetic screenings.

What is CRISPR screening?
CRISPR screening is a large-scale genetic loss-of- or gain-of-function experimental approach to find key genes that are important in biological processes. For example, it can be used to identify genes that are causing cells to be either resistant or sensitive to a drug or to identify novel vulnerabilities to target in cancer.

The concept behind the loss-of-function CRISPR screening involves knocking out every potentially significant gene by targeting only one gene per cell. The aim is to generate a heterogeneous population of cells with a different gene knocked out in each cell. In pooled screening experiments, these cells with various genetic alterations are combined in the same culture dish. While some cells may die, others will survive and become the dominant cell type. Following a few days of culturing the knockout cells, next-generation sequencing (NGS) is performed to analyze the entire mixed cell population, revealing which genetic sequences are present, depleted, or absent. Such experiments identify genes that are crucial for cell survival under normal conditions or under specific conditions, like drug exposure or other physiological scenarios.

Which library?
Currently, there are libraries designed to cause three types of gene expression modification: knockout, repression (CRISPRi), and activation (CRISPRa) which should be chosen based on the biological question. The most common CRISPR sgRNA libraries used for an in vitro screening are the genome-wide knockout libraries generated by the Sabatini/Lander lab and the Zhang lab (Wang et al. 2014, Sanjana et al. 2014).

Types of CRISPR screens and delivery of sgRNAs
CRISPR screens can be conducted either in pooled or arrayed formats. In pooled CRISPR screens, a library of single-guide RNAs (sgRNAs) is introduced into a large population of cells, where each cell experiences a unique sgRNA-mediated genetic alteration. Typically, lentiviral or retroviral vectors are used to deliver sgRNAs, which integrate into the DNA of cells already expressing the Cas9 enzyme. This allows for the identification of individual cell's genetic perturbations through high-throughput sequencing of the sgRNA sequences. To ensure that each cell is infected with only one virus and thus contains only one perturbation, a low multiplicity of infection (MOI) is often used, typically around 0.3. However, infecting only 30% or less of the cells means that a larger number of cells are required beyond those that are infected. Moreover, it's crucial to infect at least 500 cells per targeting sequence to ensure sufficient coverage (500x coverage) and obtain results within the sensitivity of the assay. For instance, if a CRISPR library contains 100,000 targeting sequences, it is recommended to use at least 1.67 x 10^8 cells. CRISPR screens can additionally be performed using arrayed sgRNAs, testing single or a small number of sgRNA(s) per well in one or more multi well plate. Although arrayed screens are preferred when having an already short-listed genes to study or for validation, they are low throughput and less cost-effective than pooled screens.

Control sgRNAs play a vital role in any screening experiment. It is crucial to include a sufficient number of negative control sgRNAs in each screen, which are expected to have no biological effect. Positive controls are also highly recommended whenever they are available.

Conclusion
CRISPR screens offer great potential for uncovering genes and genetic sequences having role in various physiological pathways and pathological conditions. While it is mostly used in in vitro screens, in vivo CRISPR screens are also emerging mostly in the field of cancer biology and hopefully will give more insights to the researchers.

References
Holcomb, E.A. et al. (2022) ‘High-content CRISPR screening in tumor immunology’, Frontiers in Immunology, 13. doi:10.3389/fimmu.2022.1041451.
Johannesen, H.J. and Netanya Y Spencer, M. (2022) Introduction to CRISPR screening: IDT, Integrated DNA Technologies. Available at: https://eu.idtdna.com/pages/education/decoded/article/overview-what-is-crispr-screening (Accessed: 28 April 2024).
Miles, L.A., Garippa, R.J. and Poirier, J.T. (2016) ‘Design, execution, and analysis of pooled in vitro CRISPR/Cas9 screens’, The FEBS Journal, 283(17), pp. 3170–3180. doi:10.1111/febs.13770.
Poirier, J.T. (2017) ‘CRISPR libraries and screening’, Progress in Molecular Biology and Translational Science, pp. 69–82. doi:10.1016/bs.pmbts.2017.10.002.
Sanjana, N.E., Shalem, O. and Zhang, F. (2014) ‘Improved vectors and genome-wide libraries for CRISPR screening’, Nature Methods, 11(8), pp. 783–784. doi:10.1038/nmeth.3047.
Wang, T. et al. (2014) ‘Genetic screens in human cells using the CRISPR-Cas9 system’, Science, 343(6166), pp. 80–84. doi:10.1126/science.1246981.
czosa MK, Mecsas J. (2016). Klebsiella pneumoniae: going on the offense with a Strong Defense, Microbiol Mol Biol Rev 80:629 - 661. https://doi.org/10.1128/MMBR.00078-15.