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Perturb-Seq Method

What is it?

The Perturb sequencing method is a technology used to observe the function of many different genes in various cells to investigate gene function on a larger scale. This method allows scientists to surpass the limitations by increasing genetic screens' sensitivity and scale by orders of magnitude.

There are two main steps: the first being to perturb a set of target genes using CRISPR technologies to alter a cell's genome. CRISPR technologies are developed from a bacteria and archaea defence system which contains CRISPR sequences. The second step is to determine how perturbation, or precise changes, of the targeted genes, affect other genes' pattern, which is done from single-cell RNA sequencing providing a list of gene expression from single cells. With the use of computers, scientists can read the tags attached to the mRNA sequences and put together mRNA identities from each cell. This further allows researchers to evaluate gene expression profiles to observe how different each cell is from one another.


Image courtesy of Princeton University.


How has it been applied thus far?

Scientists at Harvard and MIT have applied this method to genes associated with ASD or autism spectrum disorder on a developing mouse brain. Paola Arlotta, the co-senior author of the Golub Family Professor of Stem Cell and Regenerative Biology at Harvard, describes that many "risk genes" have been identified to be "associated with the development of autism spectrum disorder." This is the first time the Perturb-Seq method has been applied to a developing organism. "The challenge in the field," Artotta explains, "has been to make the connection between knowing what the genes are, to understanding how the genes affect cells.." The purpose of this was to see how the mutations impact the cell types; humans can benefit from this by better understanding complex disorders.


The researchers combine CRISPR and Cas9 gene editing to make perturbations in 35 distinct genes linked to the increased risk of developing ASD. They then used single-cell RNA sequencing to see how the gene expression changed on over 40,000 individual cells. This allowed the researchers to see how risk genes affected the brain's cortex. To prove the method's accuracy, they did a comparison with post mortem human brains with ASD and concluded that the same types of genes were also affected in the Perturb-Seq data.


Image courtesy of Paola Arlotta labroratory/Harvard University.


Does this Method Have Potential?

With this sequencing method, researchers can better understand ASD and different psychiatric disorders, and from that data, create treatments. "Our work also paves the way for Perturb-Seq to be applied to organs beyond the brain, to enable scientists to understand better the development or function of different tissue types as well as pathological conditions," says Arlotta. Since the method is also applicable to other organs, scientists in the future can apply this method to observe a range of different diseases from a genetic level. One of the main benefits described by Feng Zhang, an investigator at the MIT's McGovern Institute, is that instead of having to analyze each gene, others can "screen all of these genes in animal models in a much more efficient manner enabling us to understand mechanistically how mutations in these genes can lead to disease."


References


Blog, R. (2020, May 14). Direct capture perturb-seq – CRISPR Meets single-cell RNA sequencing to offer unprecedented detail and depth of insight from such genetic disruption studies: Rna-seq blog. Retrieved February 07, 2021, from https://rna-seqblog.com/direct-capture-perturb-seq-crispr-meets-single-cell-rna-sequencing-to-offer-unprecedented-detail-and-depth-of-insight-from-such-genetic-disruption-studies/

Lau, J. (2020, December 02). Researchers investigate autism spectrum disorder genes. Retrieved February 07, 2021, from https://news.harvard.edu/gazette/story/2020/12/researchers-investigate-autism-spectrum-disorder-genes/

Schraivogel, D., Gschwind, A.R., Milbank, J.H. et al. Targeted Perturb-seq enables genome-scale genetic screens in single cells. Nat Methods 17, 629–635 (2020). https://doi.org/10.1038/s41592-020-0837-5



Article Author: Idil Gure

Article Editors: Victoria Huang, Maria Giroux

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