Discussion
The DNA editing strategy based on Cas9 is the most widely used in gene expression manipulation. However, it cannot achieve temporary or reversible gene expression modification. The existence of the off-target effect can also cause unpredictable permanent changes to the genome. RNA-targeted gene knock down has become an option in many application scenarios. Cas13b and Cas13d are currently the most well-studied RNA editing systems, but the comparison between the two systems for their efficiency in vitro and in vivo needs further investigation.
The selection of gRNA is an important factor affecting the efficiency of CRISPR system. Using firefly luciferase as the target we systematically studied the sequence and length of gRNA on RNA degradation efficiency of Cas13b and Cas13d. We used three strategies to design gRNAs for luciferase RNA knock down. 33 gRNAs were tested with two Cas13 system. Our results indicated that the RNA degradation efficiency of Cas13 proteins on target molecules varies with different gRNA sequences, however we haven’t found significant difference between the three strategies including the stragegy of randomly selection.
Two predict tools were published for gRNA selection (cas13design[32] and CASowary[33]). We used them to valuate the 33 gRNAs. It seems that the prediction results vary with the different methods. Of the 8 gRNAs that ranked in the top 25% in our study, cas13design scored 2 of them in the top 25% of predictions, and CASowary did not rank any of them as the best grade; of the 17 gRNAs that ranked in the bottom 50% in our study, cas13design rated 9 of them in the top 50% of predictions, while CASowary rated 5 of them in the top 50%. Futhermore, the score distribution of the highest grade gRNA predicted by CASowary is not consistent in cas13design, 27 gRNAs of the 70 best graded gRNAs in CASowary were rated in the bottom 50% by cas13design (Fig.S5). There is also no obvious relationship between GC content and the efficiency according to the data of the result of the 33 gRNAs against the luciferase RNA. We also do not find any sequence preference for the flanks or ends of gRNAs, of the 33 gRNAs for luciferase, 14 had PFS with NAN/NNA, but only 4 of these gRNAs in Cas13d and 8 in Cas13b ranked in the top 50% for efficiency, which does not agree with those proposed in the literature that 3’ends of non-G or NAN/NNA forms favors RNA degradation [37, 38]. It is worth noting that the gRNAs targeting the middle part of the luciferase RNA sequence has a better effect on average. The similar results are also obtained from the research on endogenous genes Zeb1 and Dnmt3a . These results confirmed the previous finding that highly efficient gRNA is generally located in the intermediate region of the transcript, especially in the 30%-70% length position[33]. And a slight decrease of gRNA efficiency was also observed in the range of 50 nt near 5 ’downstream and 3’ upstream that was reported before[32]. It is true that the gRNAs were concentrated in the middle of the mRNA when using the CASowary algorithm (Fig.S5B). So, a comprehensive consideration of various factors can improve the possibility of selecting high-efficiency gRNA.
Previous studies have shown that 24–30 nt is required for guide-target duplex formation of Cas13[32, 37]. In our study, when the length of gRNA is 30 nt, the RNA cleavage efficiency of Cas13d is higher than that of Cas13b. While Cas13b performs better under shorter gRNA conditions.
Cas13b and Cas13d have the ability to process precursor crRNA to generate mature crRNAs[23][16]. We compare the ability of Cas13b and Cas13d to cleave precursor crRNAs for gene knock-down, and the results show that both proteins can process precursor crRNAs into mature crRNAs, but Cas13b seems to be more efficient.
Our study shows that both Cas13b and Cas13d have certain off-target potency as previous research[39], and the off-target degree of the two is similar under similar targeting efficiency. A recent study also shows that Cas13d has a high off-target rate in Drosophila cell lines and some human cell lines except HEK293T [24]. We found that there was no strong relationship between the differential gene sequence and the gRNA sequence as a whole (Fig, S4). So the existence of off-target effect might be from the collateral activity of the Cas13 proteins which has been used to develop a sensitive method for detection of a variety of viruses and nucleic acids[9, 40-45]. Unlike DNA editing systems, RNA editing does not elicit irreversible damage to DNA molecules in cells, and can restore RNA levels after treatment cessation (Fig, S3).
The feasibility of the application of Cas13b and Cas13d in vivo has not been well studied. Our work shows that the Cas13b expression system inserted into the mouse Rosa26 locus can effectively inhibit the activity of luciferase as a transgene endogenously expressed in mice. Cas13b or Cas13d transgenic mice targeting mCherry RNA can inhibit the expression of mCherry in mouse liver, wherein the mCherry expression plasmid is exogenously injected by hydrodynamic injection method.
In order to explore whether Cas13b or Cas13d can be used for intervention of endogenous gene function, we inject Cas13b(d)/gRNA expression plasmid targeting NF-kB or TNFa into mice through tail vein by hydrodynamic injection method. The results show that it can successfully alleviate LPS/D-GalN-induced acute liver failure in mice. These results provide experimental basis for the application of Cas13b(d)/gRNA system in the study of mouse gene function or clinical disease intervention.
In this study, we systematically compare the RNA degradation efficiency of Cas13b and Cas13d in vitro and in vivo. Both Cas13 systems have been proved to be a good alternative to RNAi to regulate gene expression at the RNA level without damaging the integrity of the genome. However, the factors affecting the off-target rate of Cas13 system and the duration of its action in mammals after delivery remain to be further explored.