Programmable RNA Targeting Using CasRx in Flies

Abstract

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

“…Ideally, to maximize all the capabilities of SHERLOCK and expand the CRISPRDx toolkit, it is important to evaluate alternative Cas enzymes that can complement or supplement the system. Similar to Cas ribonucleases used in other CRISPRDx systems, Cas13d enzymes such as RfxCas13d (CasRx), exclusively target RNA species that trigger subsequent collateral cleavage of bystander RNA [44][45][46] . Collateral cleavage is initiated following on-target ssRNA cleavage by the HEPN domain-based endoRNase heterodimer, which activates trans-cleavage of nonspecific bystander RNAs 20,44,46,47 .…”

“…Furthermore, Cas13d enzymes are approximately 20% smaller than Cas13a-Cas13c effectors, and do not require a Protospacer Flanking Sequence (PFS) 20,44,46,48 , presenting an advantage for protein production and flexible targeting. While the genetic modulatory effects of CasRx have been thoroughly characterized in Drosophila, zebrafish, and human cells 44,45,49 , and its putative prophylactic properties against SARS-CoV-2 have been demonstrated 40 , its potential as a diagnostic system has not yet been explored.…”

“…1A). Following on-target cleavage, CasRx exhibits collateral cleavage of off-target nucleic acids 44,45 , a feature shared by Cas nucleases used in other CRISPRDx systems [22][23][24][25] . We exploit the collateral cleavage activity of CasRx to detect SARS-CoV-2 in both synthetic templates as well as in patient-derived samples via fluorescence-based readout, and paper-based lateral flow assay.…”

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways

“…Mosquito researchers can be envious of Drosophila ’s genetic tools, such as unparalleled annotation and manipulation of its genome [ 277 ], plethora of genetic markers and balancer chromosomes, and binary transgenes to trigger RNAi and CRISPR genome editing [ 278 , 279 ]. We envision that more and more investigators like in the Akbari and Lau labs [ 234 , 280 , 281 , 282 , 283 ] and other notable Drosophilists [ 284 , 285 ] will leverage their initial foundation of studies in Drosophila and extend their expertise to the mosquitoes.…”

Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

“…Previously, we have described a set of optimal design rules for RfxCas13d guide RNAs (gRNAs), and developed a computational model to predict gRNA efficacy for all human protein-coding genes 5 . However, there is a growing interest to target other types of transcripts, such as noncoding RNAs (ncRNAs) 6,7 or viral RNAs 8,9 , and to target transcripts in other commonly-used organisms [10][11][12][13] . Here, we predicted relative Cas13-driven knock-down for gRNAs targeting messenger RNAs and ncRNAs in six model organisms (human, mouse, zebrafish, fly, nematode and flowering plants) and four abundant RNA virus families (SARS-CoV-2, HIV-1, H1N1 influenza and MERS).…”