Article

Conversion of IscB and Cas9 into RNA-guided RNA editors

RNA plays a central role in biology as it bridges the flow of genetic information from DNA to proteins. Significant effort has been devoted to developing therapeutics that alter the expression level, splicing pattern, or sequence content of mRNA. Because the consequence of RNA editing is not heritable, inadvertent off-targeting events will not result in heritable genetic scars. When combined with long-persistence delivery tools such as adenovirus-associated virus (AAV),¹ a single delivery of RNA editor can lead to a long-lasting impact.²

Tools developed from the Cas9 family of RNA-guided DNases have revolutionized genomic medicine.^3,4,5 Serving as the effector of the type II CRISPR-Cas system, Cas9 utilizes the associated crRNA/tracrRNA as the guide, opens R-loop at the matching DNA target that is flanked by an interference protospacer-adjacent motif (PAM), and introduces a double-strand break via its HNH and RuvC nuclease domains.^{6,7,8,9,10,11} It was later discovered that Cas9 originated from IscB, an RNA-guided homing nuclease in the IS200/605 family of transposons.^12,13 The associated OMEGA RNA (ωRNA) is a natural fusion of crRNA and tracrRNA, twice their combined size, and plays additional roles than driving ribonucleoprotein (RNP) assembly and serving as the guide.^13,14 IscB protein is one-third or half the size of a typical Cas9 and lacks the REC domain in Cas9. Nonetheless, it adopts a similar overall architecture to Cas9, searching for and cleaving at the DNA target using the same mechanism (Figure 1A).^15,16,17 Even the built-in allosteric motion in the HNH nuclease domain is conserved between IscB and Cas9.^{17,18,19,20,21} IscB’s compact size and tunable DNA nicking activities make it a desirable platform for CRISPR 2.0-type genome editing applications, such as base editing and prime editing.^22,23 While the naturally occurring IscBs have marginal editing efficiency in human cells, we and others showed that the engineered versions can achieve much higher insertion or deletion (indel) and base editing efficiencies.^24,25,26

Many DNA-targeting CRISPR effectors, including Cascade, Cas9, and Cas12f, have been shown to also bind single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) in an RNA-guided fashion.^27,28,29,30 Some Cas9 homologs were shown to be further capable of ssRNA cleavage.^31,32,33 This activity in some, but not all, cases requires the assistance of a PAMer ssDNA oligo (i.e., SpCas9).^27,31 A dead version of SpCas9 was named RCas9 and utilized for RNA tracking or transcript knockdown.^34,35 Cas7-11, the type III-D CRISPR-Cas effector, was also shown to be capable of RNA targeting in human cells.³⁶ However, neither RCas9 nor Cas7-11 gained popularity in in vivo applications, presumably because their performance was not robust. The type III effectors have also been utilized for in vivo RNA knockdown in human cells^37,38; however, the multi-component systems are cumbersome to use. Most researchers chose Cas13 from type VI CRISPR systems for RNA-guided RNA-editing applications.^{39,40,41,42,43,44,45,46,47} However, the majority of Cas13s further cleave the bystander mRNAs, and this collateral damage was shown to be quite toxic to bacterial and mammalian cells.^42,48 In fact, cytotoxicity was the main reservation for moving Cas13-based therapeutics into clinical trials.

In this study, we show that IscB can be converted into an exclusive RNA-targeting tool when its target-adjacent motif (TAM) interaction domain (TID) is deleted. The resulting R-IscB mediates a robust and versatile set of RNA-guided RNA-targeting applications in eukaryotic cells, including splicing perturbation, mRNA cleavage, RNA-guided A-to-I editing, and RNA-guided trans-splicing. The former two activities can be used to target dominant-negative genetic diseases, whereas the latter two can find usage in curing recessive genetic diseases through sequence correction at the mRNA level. R-IscB is more active than Cas13 and has no discernible cytotoxicity in human cells. We further show that four different Cas9s can be converted to an exclusive RNA-targeting enzyme following the same approach. These tools are attractive alternatives to Cas13 in research and medicine.