In the last one decade, CRISPR has been evolving into a pivotal technology for crop improvement, functional genetics, and biomedicine, including disease diagnostics and gene therapy. However, all currently available CRISPR tools are used to edit a DNA or RNA sequence, which may have side effects, including off-target impact. A recent study, published in Science by Hu et al. reported a novel CRISPR/Cas system, CRISPR/Cas-III-E (Craspase) (Hu et al. 2022). Craspase is a dual CRISPR gene editing system that functions at both RNA and protein levels (Hu et al. 2022). Craspase has minimal to no cytotoxic and off-target effects, which is invaluable in both basic and applied research, including gene therapy applications.

CRISPR/Cas system has quickly expanded in the last one decade

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas) system is a natural adaptive immune defense system; many bacteria and archaea use it against foreign genetic element invasions, such as bacteriophages (van Beljouw et al. 2023). Since it was recognized as a genome editing tool in 2012 (Gasiunas et al. 2012; Jinek et al. 2012), CRISPR/Cas system and its applications have been attracting unprecedented attentions from both academic and industrial communities, including pharmaceutical and biomedical companies. Due to its versatile functions, CRISPR/Cas has been reengineered into different genome editing tools (Li et al. 2023). These engineered CRISPR/Cas tools have been widely used to monitor and edit DNA/RNA sequencing for both basic biological and applied biomedical research, including drug screening, disease diagnostics and treatments (Rodrigo et al. 2023). To expand its usage, scientists have been continuously exploring new CRISPR/Cas systems while also modifying current CRISPR/Cas systems (Makarova et al. 2020). Presently, hundreds of CRISPR/Cas systems have been identified (such as Cas9 and Cas12) and/or optimized (Makarova et al. 2020), such as SpRY (Walton et al. 2020; Zhang and Zhang 2020). Based on distinct features of Cas components and the sequence similarity and phylogenetic analysis of Cas proteins, current CRISPR/Cas systems can be divided into 2 classes (Class 1 and 2), 6 types (Type I to XI), and 33 subtypes (Makarova et al. 2020). The major difference between the Class 1 and the Class 2 systems is that the Class 2 CRISPR/Cas system has only one large Cas protein for binding and degrading target sequences, while the Class 1 CRISPR/Cas systems usually have multiple Cas proteins and these proteins need to form a functional complex to bind and act on their targets. From the engineering standpoint, the Class 2 CRISPR/Cas system is much easier to be reconstructed and transformed into target cells. Currently available CRISPR genome/gene editors use the Class 2 CRISPR/Cas systems (Li et al. 2021). Based on its origin and function, Class 2 CRISPR/Cas systems are further divided into different types. Among these types, Cas 9 from Type II, Cas13 from Type VI, and Cas12 from Type V are widely used for biomedicine-related study and application nowadays. In contrast, due to its complexity, no single CRISPR/Cas system from the Class 1 has been reprogramed for biomedical engineering purpose. A recently published study in Science revealed that this newly identified Class 1 Type III-E is an atypical CRISPR Type III system, in which there is one single protein, termed giant Repeat-Associated Mysterious Protein (gRAMP), which plays the same role as the Class 2 Cas and the other Class 1 multiple protein complexes. gRAMP is a fusion protein containing four Cas7-like domains, one Cas11-like domain, and a big insertion domain (BID) (Hu et al. 2022; Liu et al. 2022; Wang et al. 2022). This makes the Type III-E Cas7-11 possible to reconstruct for the bioengineering purpose. More importantly, this paper by Hu et al. demonstrated for the first time that Type III-E Cas7-11 functionally edited a gene at both RNA and protein levels. This finding will greatly advance the CRISPR genome editing field because it significantly reduces the off-target impact and cytotoxicity, two major factors limiting CRISPR/Cas application in biomedicine (Hu et al. 2022).

Craspase is a dual CRISPR gene editor

Type III-E Cas7-11 system has a unique protein gRAMP; gRAMP is frequently associated with tetratricopeptide repeat (TPR)-Caspase HetF related to the TPRs (CHAT), a caspase-like protein with N-terminal TPRs. Caspases are a family of cysteine proteases; thus, gRAMP(Cas7-11)-TPR-CHAT CRISPR III-E is also called CRISPR-guided caspase (Craspase) (van Beljouw et al. 2021). They elucidated that Craspase plays dual functions of endonucleases and proteases through a perfect target-paired RNA-mediated allosteric regulatory mechanism (Hu et al. 2022). By using cryo-electron microscopy (cryo-EM) and biochemical engineering techniques, Hu et al. discovered that the gating loop, the linker between Cas7.2 and Cas11 domains, plays an important role in controlling Cas7-11 RNase activity and TPR-CHAT protease activity (Hu et al. 2022). Cas7-11 cleaves a targeted RNA sequence into three parts at two specific sites [site 1 at the 3rd and site 2 at the 9th nucleotide (nt) positions) that are 6 nt apart (Hu et al. 2022; van Beljouw et al. 2021). During the resting state (apo), N-terminal region of the gating loop sits in the site 1 position and thus sterically blocks gRAMP function. When a target RNA loads onto the gRAMP, the target RNA binds to the crRNA spacer segment and allows the gating loop to release the RNase catalytic activity at the site 1. In both resting and self-RNA-bound states, the CHAT protease activity was inhibited by an autoinhibited conformation (Hu et al. 2022). Non-self-RNA [containing a non-matching protospacer flanking sequence (PFS)] induces conformational changes in the CHAT domain, and as the result, the cleft between gRAMP and TPR-CHAT widens, which allows the peptide substrate access to the binding surface and finally initiates the protein cleavage (Fig. 1A) (Hu et al. 2022).

Fig. 1
figure 1

The mechanism of Craspase-targeting gene regulation and its potential application in gene therapy and disease diagnostics. (A) The model of Craspase as a dual CRISPR gene editor. Through RNA sequence complementary and structure change, Craspase switches the function for protein or RNA cleavage. This figure was adopted and modified from Hu et al. [3]. (B) Craspase can directly target mRNAs or protein sequences for gene therapy. There are many genes associated with different diseases and through targeting these genes for cleavage at either mRNA or protein levels, Craspase can more accurately edit a disease-causing gene without off-target impact. Craspase can also be modified to become a deactivated Craspase (dCraspase), in which it still binds to a specific RNA and/or protein sequence but does not exhibit the cutting function. In this way, dCraspase can be used to detect and monitor a specific disease-causing gene or virus RNA sequence for diagnosing specific diseases or viruses. dCraspase also can be linked to a specific functional enzyme, such as protein phosphatase or diphosphatase, for modifying protein function. This figure was prepared by using BioRender

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By the RNA-guided structure changes, Craspase perfectly controls crRNA-guided and non-self-RNA-dependent activities of Cas7-11 RNase and TPR-CHAT protease, and allows the cleavage of RNAs or proteins in different situations (Fig. 1A) (Hu et al. 2022; Liu et al. 2022; Wang et al. 2022). Another study also showed that structure-guided engineering of Cas7-11 can be compacted into single-vector AAV packaging for transcript knockdown in human cells (Kato et al. 2022). This suggests that Cas7-11/Craspase can be used for in vivo editing of RNAs and/or proteins for various biologic and biomedical purposes, including crop improvement and gene therapy (Fig. 1B).

Craspase has great application potentials in gene therapy and biomedicine

Ever since CRISPR/Cas was recognized as a genome/gene editing tool, its applications on human disease diagnostics and treatments have been a hot research field and significant progress has been made in the past 10 years. Many CRISPR/Cas gene therapies have been used in some clinic trials to treat human diseases, such as genetic disorders and cancers (Zhang 2021). However, the therapy precision and safety are two major concerns that limit its usability. Off-target editing frequently occurs during CRISPR/Cas9 genome editing, the most widely used CRISPR/Cas system in gene therapy clinic trials and fundamental biomedical research (Table 1). Several studies have also shown CRISPR/Cas9 genome editing causes unexpected DNA mutations, including big DNA fragment deletions and large DNA structural variants at both on-target and off-target sites (Höijer et al. 2022), which could result in serious consequences, such as cell death and other genetically inherited problems (Höijer et al. 2022). The study by Hu et al. demonstrated that Craspase can target on both RNA and protein sequences instead of DNA sequences which could minimize the inheritable problems. Furthermore, Craspase has an additional mechanism to guide RNA/protein sequence targeting which could minimize the off-target effects (Hu et al. 2022). These two regulatory mechanisms allow Craspase to target a gene sequence more accurately with much less, or no, off-target effects. The study also showed that Craspase does not cause collateral RNA cleavage; thus, Craspase does not cause cytotoxicity in eukaryotic cells (Hu et al. 2022; Özcan et al. 2021). Given that cytotoxicity and immunotoxicity are two common concerns in using the CRISPR/Cas system in gene therapy, Craspase has great advantages over currently available CRISPR genome editing tools, such as CRISPR/Cas9 and CRISPR/Cas13 (Table 1; Fig. 1B).

Table 1 Comparison of Craspase with other three commonly used CRISPR/Cas genome/gene editors
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Craspase can not only be used to target a specific RNA and/or protein for CRISPR gene therapy, but also has the potential to diagnose and treat infectious diseases (Fig. 1B), such as HIV and COVID-19, similarly to Cas9/Cas12 (Abavisani et al. 2023; Zhou et al. 2022). By deactivating Craspase through removing its RNase and protease activities but keeping its binding functions, it can be used for imaging of targeting RNA and protein sequences in human cells, particularly pathological cells such as cancer cells (Fig. 1B), which will greatly enhance our understandings of molecular mechanisms of various human diseases.

Concluding remarks and perspectives

Craspase is the first CRISPR/Cas system to exhibit the protein editing function and is also the first CRISPR/Cas system with dual functions in editing both RNA and protein. This new system has great applicative potentials in both foundational and applied biomedical research, which could revolutionize CRISPR gene therapy and human disease diagnostics as well as gene functional study and crop improvement. Particularly as the quick development of genome sequencing (Liu and Zhang 2022; Pareek et al. 2011), more and more new genes/sequences have been identified, CRISPR/Cas genome editing, including Craspase provides a powerful tool for studying gene function and its application. In the next few years, more research may focus on developing different genetic and epigenetic Craspase tools for precisely targeting etiological genes and a reliable delivery system for Craspase, which is necessary for precise gene therapy.