Thanks to CRISPR-based genetic screens, researchers were able to detect genes that play a major role in lung cancer metastasis, cancer immunotherapy, sickle-cell anemia, and a host of other medical disorders.
Targeting RNA: Cas13 enzymes traveling along an RNA landscape. Artist: Christian Stolte; co-first authors, Hans-Hermann Wessels and Alejandro Méndez-Mancilla, and study senior author Neville Sanjana (l-r, bottom). Image Credit: New York Genome Center.
But CRISPR-based genetic screens are known to have limited scope—they can only target or edit DNA. In the human genome, many of its regions that target DNA may be ineffective, and currently, available DNA-targeting CRISPR screens cannot be used to target other microorganisms, for example, RNA viruses like flu or coronavirus.
Now, in the latest, significant resource intended for the scientific community, scientists in the laboratory of Neville Sanjana, Ph.D., at New York University and the New York Genome Center have identified a new way to target the RNA by creating a novel type of CRISPR screening technology. The study was recently published in the Nature Biotechnology journal.
The scientists exploited Cas13, a newly defined CRISPR enzyme that targets RNA and not DNA. With the help of the Cas13 enzyme, the researchers designed an improved platform for largely parallel genetic screens at the level of RNA in human cells.
With the help of this screening technology, several aspects of RNA regulation can be interpreted and the function of non-coding RNAs can be identified. Non-coding RNAs are RNA molecules that are generated but do not code for proteins.
By focusing on an unlimited number of different sites in human RNA transcripts, the scientists successfully built a machine learning-based predictive model to speed up the detection of the most effective Cas13 guide RNAs.
Scientists can access the new CRISPR screen technology through an open-source toolbox and interactive website to predict the efficiencies of guide RNAs for customized RNA targets. Pre-designed guide RNAs are also provided by the new technology for all human protein-coding genes.
We anticipate that RNA-targeting Cas13 enzymes will have a large impact on molecular biology and medical applications, yet little is known about guide RNA design for high targeting efficacy. We set about to change that through an in-depth and systematic study to develop key principles and predictive modeling for most effective guide design.”
Dr Neville Sanjana, Study Senior Author, New York Genome Center and New York University
Dr. Sanjana is also a Core Faculty Member at the New York Genome Center, an Assistant Professor of Neuroscience and Physiology at NYU School of Medicine, and an Assistant Professor of Biology at New York University.
Cas13 enzymes are essentially Type VI CRISPR, short for clustered regularly interspaced short palindromic repeats, enzymes that have lately been discovered as programmable RNA-targeting, RNA-guided proteins with nuclease activity that enables target gene knockdown but without modifying the genome.
Such a trait renders the Cas13 enzyme as a promising and important therapeutic for impacting the expression of genes without permanently modifying the sequence of genomes.
This is the kind of technology innovation that we foster and develop at the New York Genome Center. This latest CRISPR technology from the Sanjana Lab has exciting implications to advance the fields of genomics and precision medicine.”
Tom Maniatis, PhD, Evnin Family Scientific Director and Chief Executive Officer, New York Genome Center
Hans-Hermann Wessels, a postdoctoral scientist, and Alejandro Méndez-Mancilla, a Ph.D. student, both co-first authors of the study, created a set of new Cas13-based tools and performed a transcript tiling as well as permutation screen in mammalian cells. On the whole, the scientists collected data for over 24,000 RNA-targeting guides.
“We tiled guide RNAs across many different transcripts, including several human genes where we could easily measure transcript knock-down via antibody staining and flow cytometry,” stated Dr. Wessels. “Along the way, we uncovered some interesting biological insights that may expand the application of RNA-targeting Cas13 enzymes.”
For instance, one of the findings made by the researchers provided a better understanding of the type of regions of the guide RNA that are more significant for detecting a target RNA. The researchers used scores of guide RNAs with 1, 2, or 3 single-letter mismatches to their intended RNA and detected a crucial “seed” region that is remarkably susceptible to mismatches that exist between the target and the CRISPR guide.
This finding will help researchers develop guide RNAs to prevent off-target activity on unplanned target RNAs. Considering that a normal human cell expresses roughly 100,000 RNAs, precise targeting of the only intended target, that is, the Cas13 enzyme is crucial for both therapeutic and screening applications.
Apart from advancing one’s understanding of Cas13 off-targets, the “seed” region can potentially be utilized for state-of-the-art biosensors that can differentiate between closely associated RNA species more accurately. On the whole, this research raises the number of data points from earlier Cas13 research made on mammalian cells by over two orders of magnitude.
We are particularly excited to use the optimized Cas13 screening system to target noncoding RNAs. This greatly expands the CRISPR toolbox for forward genetic and transcriptomic screens.”
Alejandro Méndez-Mancilla, Study Co-First Author and PhD Student, New York Genome Center
In this work, the scientists observed a major variation in protein knockdown when they were targeting different protein-coding as well as non-coding elements of messenger RNAs, and discovered proof that the Cas13 contends with other RNA-binding proteins that are implicated in transcript processing and splicing.
The guide RNA predictive model was recently manipulated by scientists for a specifically crucial analysis—the public health emergency relating to COVID-19 has been caused by a coronavirus, in which an RNA, and not DNA, genome is present.
Using the model obtained from their largely parallel screens, the investigators were able to identify optimal guide RNAs that can potentially be used for upcoming detection and therapeutic applications.
Moreover, predictions for Cas13 guide RNAs for the SARS-CoV-2 strain isolated in New York are now available at http://bit.ly/coronavirus-guides.
Other coauthors of the study include Mateusz Legut, Ph.D., and Zharko Daniloski, Ph.D., and NYU Biology Ph.D. student Xinyi Guo.
Wessels, H.-H., et al. (2020) Massively parallel Cas13 screens reveal principles for guide RNA design. Nature Biotechnology. doi.org/10.1038/s41587-020-0456-9.