Introduction
Clustered regularly interspaced short palindromic repeats (CRISPRs) is a kind of short regularly spaced repeats, contain 21-37 nt direct repeats (DRs) which is widely present in prokaryotes including almost all archaea and many bacterial genomes[1]. Together with a protein family called CRISPR associated protein (Cas)[2], it forms the adoptive immune system of prokaryotes to protect them from foreign replicons[3, 4]. Four Cas variants (Class 1-4) have been found[1, 5]. After CRISPR-Cas modules were discovered, they quickly attracted great attention and have been developed into various gene editing tools. Among them, CRISPR/Cas9 system, belonging to the Class 2 type II system, is widely used for gene and cell functional researches. Recently, the attempt of using CRISPR/Cas9 system in clinical treatment is also being actively explored[6-9].
The Cas9 system targets DNA strands to generate DNA double-strand breaks (DSBs) at the site recognized by the gRNA sequence[10]. This process can induce a p53-mediated DNA damage response and result in cell cycle arrest and apoptosis[11]. The occurrence of off-targeting has also aroused the concern about the safety of clinical application of the system. Meanwhile, DNA targeting permanently changes the genome, making reversible or temporary gene expression regulation impossible. In these scenarios, the RNA ”knock-down” method may have advantages over gene editing. Recently, the most popular RNA targeting strategy is RNA interference (RNAi). However, there are still some unmet problems with RNAi including uncertain efficiency for different RNA sequences, different cell types and possible off targeting effect[12].
CRISPR-Cas13 system[13-15], the Class 2 Type VI system, contains a single-effector protein Cas13 showing the capability to cut RNA strand navigated by gRNA [16, 17]. Up to now, four types were founded in Cas13 protein families (Cas13s), VI-A, VI-B, VI-C, VI-D, called Cas13a(c2c2), Cas13b, Cas13c and Cas13d respectively[16, 18, 19]. As the smallest Cas13 effector, Cas13d received more attention than Cas13b[16, 20-22], but researches were lack of systematic comparison between the RNA editing effects of Cas13b and Cas13d up to now. Both Cas13b and Cas13d have been used as RNA editing tools in microorganism, mammalian cells, plants, zebrafish, Drosophila with high efficiency[16, 19, 23-27], they were also used for living cell RNA imaging[28], mRNA demethylation[29], m6A modification[30] and RNA detection[31].
Some methods have been developed to predict efficiency of the crRNA for Cas13s, including CHOPCHOP (for Cas9, CasX, Cas13s and TALEN), Cas13 design[32] and CASowary[33] (both for Cas13d). However, these methods still need more practical verification.
Therefore, in this study, we compared the RNA degradation efficiency of Cas13b and Cas13d, focusing on the effect of gRNA sequence, length and its position at target RNA on the cleavage activity of Cas13 proteins. At the same time, we analyzed the occurrence of off-targeting by RNA-seq. In addition, we verified the feasibility of the application of Cas13b and Cas13d in mouse models.