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.

Photo: Janet Iwasa, NIH