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CRISPRi switches off 13 genes at once

  • October 9, 2019 at 8:58 AM
  • in CRISPR

Contrary to popular notion, evolution is more of a pianist than a (blind) watchmaker: fiddles mostly with gene regulation, trying to play on genes in a better way, and to make more beautiful music (fitter, actually). We are now one step closer to evolutionary pianists, thanks to newly developed ELSA, which stands for extra-long sgRNA array.

ELSA solves the fundamental bottleneck of known CRISPR systems. As we owe CRISPR precision to its single guide RNA molecules (sgRNA), we need multiple sgRNAs to change multiple genes. However, we had a hard time stacking them together. Most elaborate approaches managed to reach the number of 4 sgRNAs.

The new method combines up to 28 sgRNAs. Key to the success lies in avoiding repetitions. A team from Penn State University designed a long stretch of DNA, which codes all parts necessary for coexpression of 28 sgRNAs, while being diverse enough to avoid problems that haunted previous attempts.

Bacterial ELSA has over 4 thousand base pairs, which cover 64 promoters, 28 sgRNA handles, 50 terminators, and 63 spacers.

Scientists tested ELSA with CRISPRi (i for interference). In short, CRISPRi is a wolf without teeth – hunts for DNA, catches it, but doesn’t cut it. Instead, it remains glued to a gene, preventing cell machinery from reaching the gene (interfering) and essentially switching the gene off. In three experiments, using 15 – 22 sgRNAs, bacteria E. coli were successfully forced to exert distinct behaviors, such as changes in metabolism or modification of stress responses. To elicit a strong response, researchers targeted every gene by 2-3 sgRNAs simultaneously, which in the biggest experiment accounted for 13 genes. Additional analysis demonstrated the presence of complicated gene networks – for instance, influencing 13 genes affected the activity of 242 other genes.

According to the publication, CRISPR experiments with ELSA can “unlock several truly large-scale applications”. One of the authors predicts with excitement:

The future is parallel and combinatorial in both experimental and computational terms! We are on our way to simultaneously edit and knockdown, potentially 1000+ genes at the same time!

Ayaan Hossain

Update (11th October): Authors of the study shared calculator, which can be used to design details of ELSA under https://salislab.net/software/design_elsa_calculator.

Publication: Reis, A. C., Halper, S. M., Vezeau, G. E., Cetnar, D. P., Hossain, A., Clauer, P. R., & Salis, H. M. (2019). Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays. Nature Biotechnology, 1-8. Doi:10.1038/s41587-019-0286-9.



Context Watch: DNA test for gayness

  • October 15, 2019 at 12:59 PM
  • in Context Watch Society
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  • An application promises to tell "how gay you are" - contradicting the very publication it was built upon.

Is there a gay gene?

This is an outdated question. Genetics – except for a few rare diseases – ditched the concept of “a gene for something”, also known as a candidate gene. Now, whether it is cancer or height, scientists are looking for complex variations of sequences in the whole genome. They hunt not only for multiple genes but also for places outside of genes, which account for regulation.

The Study

Thanks to modern supercomputers, it is possible to compare 3.6 billion-letter-length genomes of 0.5 million people, totaling 1.8 quadrillion letters. That’s what the researchers have done in the last year, with genetics of sexual orientation in mind.

The results were presented in a publication typical for the genomic field. Authors found five places around several genes, which explained “8 to 25% of variation in same-sex sexual behavior”. They discussed functional role of those genetic variants, finding for instance intriguing connection to olfactory genes. However, the publication unusually appealed to fellow scientists:

Our findings (…) underscore the importance of resisting simplistic conclusions – because the behavioral phenotypes are complex, because our genetic insights are rudimentary, and because there is a long history of misusing genetic results for social purposes.

Communication with the public was even more careful. The team crafted website exclusively to explain the discovery (geneticsexbehavior.info) and heavily engaged with media and at Twitter.

The study obviously caused a lot of buzz. First, scientific community had heated debate about methodology (example) and ethics (example). And then, the worldwide media focused entirely on the gay gene question:

Just two weeks after the headlines downplaying the role of genetics in sexual orientation, a private company developed a test that… well, exactly checks your genome to say something about your sexual orientation. Contrary to the media coverage, contrary to the scientists – someone decided to commercialize the findings.

The Company

GenePlaza – author of the app – for mere €5.50 promises to “quantify same-sex sexual behavior”. Although they warn about lack of predictions, at the same time all charts and marketing clearly point to predictions for individuals. Misuse of the science is evident, as a user sends them genetic data, and in return receives the following assessment:

Belgian company is no stranger to this odd business.

Their shop have many more misleading apps. One of them claims to check your genetic intelligence and is advertised by “did you read more books than your genes expected to” (!). Other, for just €4, links your genome to math abilities. There are also apps for depression, neuroticism, as well as classic ancestry and health-related tests.

GenePlaza was co-founded by bioinformatician Alain Coletta and developer Robin Duqué. In recent report, Coletta said:

We try to stress the fact that this is not a disposition score, it’s not a diagnostic. We hope we’re doing a good enough job but it’s not easy to do.

The Scientist

Regardless of disclaimers, the company markets their products with questions such as “how gay are you?”. The scientific evidence behind some apps is even more obscure. For instance, the intelligence app was built upon a study in which differences in intelligence were explained by genetics only in 5%.

The Reaction

Update (15th October): Authors of the original study “urged GenePlaza to take down the application immediately”.

Joseph Vitti, scientist from Broad Institute, fiercely reacted to the app. A petition to remove the app was already signed by more than a thousand people in few days – among them, some scientists heading to the annual conference of American Society of Human Genetics.

Joseph describes the GenePlaza test as both unscientific and dangerous. Why dangerous? Surprisingly, GenePlaza social media profile provides the answer:

As countries such as Uganda try to penalize gay people, the law enforcement can reach out for GenePlaza’s tool. Although that seems like a distant possibility from Belgian perspective, in reality there are still countries forcing people to exams which determine sexual history.

GenePlaza is probably aware of the controversy, as it advertised the app with a shocking ‘testimonial’ from Uganda:

Highly rural societes like where I live in Uganda, East Africa, value child labor on the farm so much that they cannot understand the relevance of non-reproductive family members or neighbors. But maybe the potential enforcement of heterosexuality against same-sex preferers, the microaggression they feel and the discrimination they suffer is crucial for their fullest intellectual and social development.

Source

That text was removed from the website.

The Science

Genetics of the human behavior is murky. Scientists are only beginning to gather initial insights into genetic variants, that define just small part of our characteristics. Research is mostly done on European populations.

As recent editorial in The Scientist concluded:

Given these shortcomings, why provide such tests at all? – At this point, we are purely focusing on the user having a positive, fun experience – we are not asking users to take any action.

Tomohiro Takano

A positive, fun experience providing wrong data.


DDR2 variants associated with Warburg-Cinotti syndrome

  • November 16, 2018 at 7:51 AM
  • in Genetic Variants
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  • Six patients with Warburg-Cinotti syndrome were found to have mutations in DDR2 gene.

Rare syndrome named Warburg-Cinotti was discovered in 2006 and recently its genetic basis have been uncovered using exome sequencing.

DDR2 gene, governing cell reactions to collagen molecules, is essential for proper fibroblast (cells synthethising collagen) function. Any alterations in a product of that gene may lead to disruptions of tissue development – as those observed in the disorder.

In addition to diagnostic approach, the study tested a possibility of treatment influencing DDR2 product. Dasatinib successfully restored some functions of fibroblast, suggesting a new therapy for Warburg-Cinotti patients.

More: “Recurrent, Activating Variants in the Receptor Tyrosine Kinase DDR2 Cause Warburg-Cinotti Syndrome”, L. Xu et al., 2018, doi:10.1016/j.ajhg.2018.10.013.