The current worldwide Coronavirus pandemic poses a serious threat to our way of life, our economy, our health, and the health of our friends and families. During these challenging times, we have truly been inspired by the reaction of the scientific research community to come together and develop rapid solutions for potential SARS-CoV-2 diagnostics and therapeutics. To date, over 45,000 scholarly articles on Coronaviruses have been posted online. As a company that provides essential tools, reagents, and services to scientists utilizing genome engineering, we at Synthego have been honored to have played a small part in the recent development of two CRISPR-based diagnostic protocols, both of which were published online and were a feature in our blog several weeks ago. It is going to take such novel ideas, and the imagination and courage of scientists, doctors, and public health officials around the world to control the pandemic. Now, our own team of scientists and engineers are thinking of ways in which we can contribute to this effort.

In today’s blog, we will report on an effort from Synthego’s bioinformatics team, led by David Conant, Ph.D. David’s goal was to generate a useful resource for both disease researchers and diagnostics developers: CRISPR-Cas13 guide RNA designs that specifically target RNA sequences in the SARS-CoV-2 virus. 

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SARS-CoV-2 Guide Sequences

The recent COVID-19 pandemic is an extraordinary situation. In these challenging times, the efforts of researchers working on therapeutics and diagnostics have been truly inspiring. To support genome engineering researchers develop rapid solutions, our bioinformatics team has generated CRISPR-Cas13 guide RNA designs that specifically target RNA sequences in the SARS-CoV-2 virus. These guide sequences will be a useful resource for researchers working in both therapeutics and diagnostics. >>>DOWNLOAD NOW

Challenges in CRISPR Targeting of SARS-CoV-2

The SARS-CoV-2 virus, which belongs to the Coronavirus family, has a single-stranded RNA genome of ~ 27-34 kilobases. This particular virus strain is novel and was first identified in 2019. There are several unknowns that we must factor in when designing targeting gRNAs for this virus; for example, we don’t yet understand the mutation rate of the virus. Critically, there isn’t yet a ready-to-use genomic reference database out there available for researchers to efficiently design guide RNAs.

In addition, CRISPR-editing of the virus, which is RNA-based, requires the use of an RNA editing nuclease. To date, several of these have been identified, but we chose to focus on Cas13a (formerly C2c2), first characterized by Abudayyeh, Gootenberg, Konermann, et. al. in the lab of Feng Zhang in 2016, and Cas13d, first characterized by Konermann et. al. in the lab of Patrick Hsu in 2018. There is still some uncertainty around guide RNA design for the Cas13 family of nucleases, including a lack of clarity around PAM sequence requirements, secondary RNA structures that may hinder guide binding, and cell-type dependent variability in Cas13 subtype activities.

Given this information, it is clear that many researchers may need to invest efforts on the bioinformatics side to identify appropriate SARS-CoV-2 RNA target sequences that are effective for CRISPR-mediated editing. Given the speed of research necessary to combat the Coronavirus pandemic, it may not be feasible for researchers to spend their time designing and testing different CRISPR guides. In order to accelerate research in this direction, Synthego’s bioinformatics team has come up with a solution that will minimize researchers’ efforts around guide design. 

Bioinformatic Design of Cas13a/Cas13d Guide RNAs Targeting SARS-CoV-2

To date, a considerable number of scientists and doctors have submitted genomic sequences of SARS-CoV-2 from patient samples on open platforms such as ViPR (Virus Pathogen Resource). These sequences exist in various states of protein coverage, and in some cases, there is sequence divergence. Of the 3233 Coronavirus/like sequences in ViPR, we pared it down to 87 strain submissions. 

Our bioinformatics team looked for highly conserved targets to design effective guides accordingly. As one can imagine, depending on the application, the considerations for guide design changes. 

For instance, in the case of diagnostics test development, researchers would need a high degree of specificity for SARS-CoV-2 sequences. So far, two potential CRISPR-based diagnostic tests have been developed to rapidly identify SARS-CoV-2, and their protocols are published online: a Cas12a DNA-targeting approach (DETECTR platform) developed by Mammoth Biosciences and a Cas13a RNA-targeting approach (SHERLOCK platform) developed in conjunction between the Feng Lab and Abudayyeh-Gootenberg Lab. Bearing both these considerations in mind, our bioinformatics team designed 50 Cas13a guide RNAs that are as selective as possible for SARS-CoV-2 (i.e., they don’t have much overlap with other Coronaviruses).

Conversely, in the case of therapeutics development, guides that cut any coronavirus RNA, not just SARS-CoV-2, might be acceptable. In that case, sequences from all different coronaviruses can be compared and most conserved sequences can be selected to make a bigger impact with CRISPR-based therapeutics. To date, two potential therapeutic applications of using RNA-targeting Cas13 nucleases have been published: first, the CARVER (Cas13-assisted restriction of viral expression and readout) approach utilizing Cas13a/b by Frieje and Myhrvold et al., in the labs of Feng Zhang and Pardis Sabeti in 2019, and the PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells) approach utilizing Cas13d by Abbott, Dhamdhere, Liu, Lin,  Goudy et al., (not yet peer-reviewed) in the lab of Stanley Qi in 2020. Both of these approaches discussed ways to kill any viruses, not just Coronaviruses, inside human cells. With this in mind, we chose to focus on Coronaviruses and potential future derivatives. Thus, our bioinformatics team designed 50 Cas13d guide RNAs that can cut RNA sequences in many different Coronavirus strains, including SARS-CoV-2 and potential future variants. 

For both sets of Cas13 guide RNAs that we designed, we also took into account any possible off-target effects in human RNA sequences, which would be especially relevant for any potential Cas13-based therapeutic. We chose to focus our designs on the major structural proteins of SARS-CoV-2, which include the orf1ab, S (spike), M (membrane), N (nucleocapsid), and E (envelope) genes. 

Takeaway

Our hope is that this set of SARS-CoV-2 and Coronavirus targeting Cas13 guide RNA designs will be useful to researchers who are seeking to develop potential diagnostics or therapeutics against the virus. By leveraging our bioinformatics capabilities, we believe that we have simplified the selection process for these targets by designing a set of guide RNAs that will effectively target these viruses. If you are interested in obtaining these sequences (50 designs for therapeutic development applications and 50 for diagnostics), you can download them here. If you are interested in having Synthego synthesize these sequences as synthetic guide RNAs in a collaborative effort, please reach out at blog@synthego.com.

This will be the first of several projects that we will embark on at Synthego in our endeavor to help scientists around the world eradicate the SARS-CoV-2 pandemic.

SARS-CoV-2 Guide Sequences

The recent COVID-19 pandemic is an extraordinary situation. In these challenging times, the efforts of researchers working on therapeutics and diagnostics have been truly inspiring. To support genome engineering researchers develop rapid solutions, our bioinformatics team has generated CRISPR-Cas13 guide RNA designs that specifically target RNA sequences in the SARS-CoV-2 virus. These guide sequences will be a useful resource for researchers working in both therapeutics and diagnostics. 

ABOUT THE AUTHOR

Kevin Holden, Ph.D.

Kevin Holden is Head of Science at Synthego in Redwood City, California. He is a senior member of a research team responsible for integrating CRISPR genome engineering workflows into novel automation platforms. In addition, he oversees academic and industrial collaborations with key opinion leaders in the CRISPR community, and leads interactions with the company’s scientific advisors, and frequently represents Synthego’s scientific interests at conferences and events. He has over 10 years of industrial biotechnology experience that includes collaborative development research in synthetic biology and genome engineering. Kevin earned his Ph.D. in Microbiology from UC Davis with an emphasis in biotechnology. While at UC Davis, he adopted a laboratory cat named “Kitty.” He is originally from the United Kingdom and immigrated to California in his youth.