CRISPR screening enables rapid and unbiased identification of genes associated with specific biological pathways. Explore how researchers are using this cutting-edge tool to shape our understanding of biology and advance medicine.

The invention of CRISPR-Cas9 genome editing technology in 2012 ushered in a new era in biology. It provides a powerful new tool for modifying DNA in a precise and directed way.

“CRISPR-Cas9 is like cutting, copying, and pasting DNA,” explains Neville Sanjana, core faculty member at the New York Genome Center and assistant professor at New York University. “But the breakthrough for this gene-editing technology is due to its programmability. The ability to program Cas9 to move to a specific location in the human genome allows us to [its use] for large-scale screening.

CRISPR screening—using the CRISPR-Cas system to edit thousands of genes and measure the functional impact of those edits in a single experiment—has become rapidly popular and productive in the biomedical research community. became a tool.

“Over the past decade, we have harnessed the power of CRISPR screening to cast a broad net of genetic hypotheses and use that broad net to identify the genes responsible for many different diseases and traits. says Sanjana.

Widespread application of unbiased screening based on gene editing has revealed new molecular mechanisms in basic biology, cancer research, immunology, neuroscience, and many other fields, opening up available opportunities for precision medicine. is open.

What is CRISPR screening?

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system consists of a Cas9 enzyme that creates double-strand breaks in DNA and a guide RNA that directs the nuclease to specific locations. genome. When cells attempt to repair the damage, repair becomes incomplete, leading to gene knockout. CRISPR screening is an application of this technology that involves delivering large libraries of lentiviral guide RNAs to large numbers of cells.

“Since each cell has different mutations in different genes, we can study the effects of unbiased, massively parallel deletion of all genes in the genome,” Sanjana explains.

Gene knockout experiments that completely inactivate the target gene are the most common type of CRISPR screening, but the core technology is to activate (CRISPRa) or silence (CRISPRi) the target gene without permanently altering the DNA. It is also adapted so that

“CRISPRa or CRISPRi uses mutated Cas9 and RNA guides that target promoter regions to activate or repress gene expression,” says Gabriel Balmus, group leader of the British Institute for Dementia at the University of Cambridge. explains.

In the pooled CRISPR screen format, target cells are challenged with selective pressure, such as drug treatment, and subsequently evaluated through analysis of sequencing data to identify genetic alterations with positive or negative phenotypic effects. The result is typically a ranked list of genes that confer susceptibility or resistance to the particular biological challenge under investigation. The final step is to validate that the observed phenotypic changes are due to the specific effect of the mutation.

“I’m a huge fan of pooled screens because they provide a very powerful research tool without the need for complex experimental infrastructure like robotics,” Sanjana says. “This allows a single experimenter to perform genome-scale screening in a relatively straightforward manner.”

In contrast, array screens are performed in multiwell plates, allowing researchers to directly view the effects of individual guide RNAs on a well-by-well basis.

Elena Navarro Guerrero, Head of Functional Genomics, Target Discovery Institute, University of Oxford, said: “But it’s more costly and time consuming than pooled screens.”

Because each mutation is predefined, arrayed screening can also be easily combined with non-DNA sequencing readouts such as imaging, proteomics, and metabolomics profiling. However, recent advances in single-cell multi-omics offer new possibilities to obtain high-content readouts such as gene expression and epigenetic state, and to characterize individual cells with unprecedented detail from pooled CRISPR screens. is open.

Identifying genetic factors of innate immunity using custom-targeted CRISPR screens

Download this application note to learn how researchers generated customized, high-quality oligos for CRISPR screening, identified many genes that influence human monocyte phagocytosis, and analyzed genome-wide Discover how you’ve avoided the cost and time impact associated with validating hits generated by screening. .

View application note

From genes to therapy

One of the areas with the greatest therapeutic potential for CRISPR screening applications is precision medicine. Identifying genes that enhance or decrease the activity of specific drugs opens the opportunity to stratify patients prior to treatment.

The first genome-wide CRISPR screen published in 2014 showed the promise of an approach to identify mutations that confer drug resistance in cancer cells. A pooled screen of 18,080 genes with 64,751 unique guide sequences in a melanoma model identified candidate genes involved in resistance to vemurafenib, a drug that targets the mutant protein kinase BRAF . The results of such studies pave the way for more detailed genetic profiling of patient tumors and help predict whether a particular treatment would be beneficial.

“CRISPR screening enables us to realize the potential of personalized medicine and to make well-informed treatment decisions. Each mutation is associated with a specific biological phenotype,” says Sanjana.

CRISPR screening can also be used to investigate the effects of changes in noncoding regions of the genome. In 2015 Sanjana was part of the team using the CRISPR guide library. BCL11A Erythroid enhancer, a non-coding genomic element involved in regulating the expression of fetal hemoglobin.

“By reactivating fetal hemoglobin, which is normally switched off in adults, it has the potential to replace adult hemoglobin in patients with certain blood disorders that affect the production of adult beta hemoglobin. It’s been speculated for a long time that there is,” says Sanjana. .

In 2021, the results of clinical trials of gene-editing therapies will be BCL11A Enhancer published. Two of her patients, with β-thalassemia and sickle cell anemia, had received infusions of blood stem cells that were genetically edited to reactivate the production of fetal hemoglobin. Over a year later, both had high levels of fetal hemoglobin expression and no longer needed blood transfusions.

“The CRISPR guide RNA used in that clinical trial came from a library we designed for screening,” says Sanjana. “It’s a really great example of

It shows the power of this approach. ”

Accelerate therapeutic drug target validation with genetically engineered cell libraries

Download this case study to learn more about the use of genetically engineered cell libraries in host-virus interaction studies, data from genome engineering screen development and CRISPR knockout screens, and the functional and clinical efficacy of validated targets. Learn about downstream assays that demonstrate relevance.

View case study

Overcoming challenges

While it is relatively easy to perform CRISPR screens in conventional immortalized cell lines, similar studies in other cell types can be more challenging.

“One of the biggest limitations is the high number of cells required, especially for pooled screens where we need at least 70-100 million cells, and we are working with limited patient samples. This can be a big problem,” he said. Navarro Guerrero. “Another issue is that some cell types, such as macrophages and neurons, are not susceptible to lentivirus infection.”

To circumvent these challenges, many researchers are using induced pluripotent stem cells (iPSCs). This may represent a more relevant patient-derived model for studying human disease. For example, Navarro Guerrero’s team recently developed a lentiviral transduction method to deliver his CRISPR/Cas9 to human iPSC-derived macrophages with near 100% efficiency.

“This opens up exciting new opportunities to study the role of macrophages in immune responses, chronic inflammation, neurodegenerative disease, and cancer progression using efficient genome-editing techniques,” says Navarro Guerrero. .

Researchers studying neurodegenerative diseases also need to find ways to mimic the effects of aging on cells so that they can be screened for phenotypic changes associated with disease pathology. For example, Balmus’ team used his CRISPR screen to investigate the underlying disease mechanisms behind ataxia telangiectasia. ATMs Gene – Expose cells to reactive oxygen species to accelerate the aging process.

“We have discovered a transcription factor gene that, when lost, rescues susceptibility. ATMs-deficient cells to reactive oxygen species,” says Balmus. I think there are.”

Choosing the right CRISPR system for your research

The applications of CRISPR gene editing technology continue to expand. Much of the versatility of the CRISPR system comes from the CRISPR-associated or Cas proteins, molecular scissors that recognize and cut specific DNA fragments. Download this application note to find out the key differences between Cas9 and Cas12 and which ones are suitable for your research.

View application note

Discover the “unknown unknown”

CRISPR screening allows researchers to rapidly identify specific genetic alterations associated with specific biological pathways or diseases in a completely unbiased manner.

“It’s a tool that can evolve in the dish,” Balmas says. “Some of the discoveries we’re making are very counterintuitive. We couldn’t have predicted them in advance.”

This technique has provided researchers with a powerful tool for investigating a variety of biological questions. For example, to analyze the underlying causes of disease or to identify genetic alterations that confer drug sensitivity or resistance.

“What I love about working in this field is that you can be amazed,” says Sanjana. “The sense of discovery we get from these high-throughput functional genomics screens is amazing. We can step into new biology every day.”