Cas13 is capable of intrinsically targeting host RNA in mammalian cells through mechanisms that were not previously understood. Through transcript-specific mechanisms that are independent of the CRISPR RNA sequence and dynamically dependent on the conformational state of Cas13, Cas13 can also cleave host RNA. This effect is demonstrated by a team of researchers primarily from Northeastern University in Shenyang, China, using multiple Cas13-family effectors encoded in one- and two-vector lentiviral systems. Furthermore, host genes implicated in viral processes and whose transcripts Cas13 targets intrinsically help limit Cas13’s lentiviral delivery and expression. These results underline the need for caution regarding intrinsic RNA targeting in Cas13-based applications and provide guidance for the appropriate use of lentiviral Cas13 systems.

The peer-reviewed research article “Intrinsic targeting of host RNA by Cas13 constrains its utility” was published in Nature Biomedical Engineering.

The type-VI CRISPR-Cas13 systems have been designed as powerful toolkits for RNA-targeting applications such as RNA knockdown, editing, imaging, detection, and gene therapy. Cas13a, Cas13b, and Cas13d are well-known and widely used Cas13-family effectors, but new research on Cas13X/Y, Cas13bt, and Cas7-Cas11 has broadened their capabilities.

To achieve site-specific RNA targeting, all Cas13 RNA nucleases require a single guide RNA (sgRNA) that consists of two main parts: a 20–30 bp target-matching spacer sequence called the CRISPR RNA (crRNA) and a short cognate direct repeat (DR) sequence. When activated by pairing the crRNA sequence with a complementary single-stranded RNA (ssRNA) target, the Cas13 effector cleaves the target RNA. Meanwhile, activated Cas13 frequently exhibits trans or collateral cleavage activity, allowing Cas13 to degrade non-target ssRNA. This side effect has been used to boost readout signals in RNA detection applications, but it may also impair the accuracy of Cas13-mediated RNA targeting and cause cellular toxicity.

Co-first authors Zexu Li, Zihan Li, and Xiaolong Cheng discovered an unusual lentiviral defect while systematically evaluating the performance and properties of three major types of Cas13-family effectors (Cas13a/b/d) under lentiviral vectors. They define this as a lentivirus failure or compromised function that limits effective lentiviral delivery and Cas13 effector expression in target cells. 

They discovered that the lentiviral defect in Cas13 systems is caused by previously unknown intrinsic host RNA targeting by Cas13 effectors. This means that it can change endogenous RNAs, such as some virus-related host genes, without needing to interact with crRNA and only with transcripts. They also saw that Cas13 can selectively cut endogenous RNA transcripts even when crRNA or crRNA:target pairing is not present. This suggests that different Cas13 states (protein along, Cas13:crRNA binary complex, or Cas13:crRNA:on-target RNA ternary complex) have different abilities and selectivity for intrinsic RNA targeting.

Endogenous RNA-targeting activity not only limits the utility of lentiviral Cas13 systems, but it can also have unexpected consequences depending on the RNA targets and target cell types. These findings necessitate careful consideration and further testing of Cas13-based tools, particularly in clinical applications. With a better understanding of the molecular mechanism, rational mutagenesis or directed protein evolution will be needed to remove such unwanted intrinsic RNA targeting while maintaining the precise on-target RNA-processing capacity of Cas13 effectors.

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