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.