Results
Effect of gRNA sequence on RNA knock-down efficiency
Schematic diagram of the binary expression system of Cas13b(d) protein
and crRNA was represented in Fig.1A. The expression of Cas13b(d) cDNA is
driven by CMV promoter, and crRNA is expressed under U6 promoter. After
the expression of Cas13b(d) and crRNA in cells, they form a complex and
recognize the mRNA strand according to the sequence of the crRNA and
cleavage the mRNA by Cas13b(d) protein[23]. In order to facilitate
the detection, we first used the firefly luciferase as the target and
renilla luciferase as an internal reference. The crRNAs complementary to
the firefly luciferase mRNA were designed and cloned into the expression
plasmid. Cas13b(d)-crRNA and luciferase expression plasmids were
co-transfected into HEK293T. The result showed that as the amount of
Cas13b(d) plasmid increased, the efficiency of firefly luciferase
knock-down by both Cas13b and Cas13d were increased until a similar
threshold reached. However, under the same amount of Cas13b(d) plasmid,
Cas13d had a higher overall efficiency than that of Cas13b (Fig.1B).
To study whether the location site on target RNA strand and the sequence
composition of crRNA has an impact on the effect of Cas13b(d) RNA
cleaving, 33 crRNAs against luciferase were designed in three ways. The
first batch of gRNA was designed based on CRISPR-Cas13 system targeting
database
(http://chopchop.cbu.uib.no).
The second was based on Primer3
(https://primer3.ut.ee) to select
sequences with high specificity. The third was selected randomly. All
these 3 batches of gRNA showed a similar average efficiency and
tendency. The results also indicated that the efficiency of Cas13b and
Cas13d varied with different crRNA. Meanwhile, a site with high
efficiency for Cas13b did not predict the same efficiency for Cas13d.
Although no obvious relationship was found between the efficiency of
Cas13b(d) and the base composition of crRNA (Fig.1E), it was worth to
note that in our Case the crRNA selected against the position close to
the middle of the target RNA generally had better degradation effect
than those near the 5’ and 3’ marginal regions for both Cas13b and
Cas13d (Fig.1C, 1D). However, the same outcome was not observed on d2GFP
RNA as previous study (Fig.S1A-S1C)[32]. Compared with Luciferase,
the d2GFP is much shorter, so whether the target RNA length and the
secondary structure of RNA affect the efficiency of Cas13b(d) needs more
research.
Effect of crRNA length on RNA knock-down efficiency
To explore whether length of gRNA could affect the efficiency of
Cas13b(d), three pairs of crRNAs with 20 nt or 30 nt in length were
chosen to target the 5’, middle or 3’of luciferase RNA respectively.
Interestingly, Cas13b performed better than Cas13d when shorter crRNAs
were used (Fig.2A-2D). To further confirm the result , crRNAs of
different lengths were used to explore the effect of the length of crRNA
on the efficiency of the two kinds of Cas13. To avoid the influence of
protospacer flanking site (PFS) , the crRNAs with either a 5′ PFS of the
original 30 nt crRNA or a 3 ′ PFS (Fig.2E) were used. As long as the
5’PFS or 3’ PFS remained unchanged, the cut efficiency were keep
relatively stable for Cas13b but not for Cas13d (Fig.2F-2G). The most
appropriate crRNA length for Cas13b was 22-25 nt. Notably, Cas13b still
played its function even when crRNA was only 15 nt in length. On the
contrary, Cas13d was dependent on longer crRNAs, when the length of
crRNA was less than 22 nt, the effect of Cas13d was largely affected.
The same tendency was obtained using another target site of luciferase
RNA which Cas13b showed better efficiency than Cas13d with 30 nt gRNA
(Fig.2H, 2I).
The crRNA precursor processing ability of Cas13b(d)
In order to test the processing ability of crRNA precursor of Cas13b(d),
crRNA sequences located in the 5’, middle or 3’ region of luciferase RNA
were selected respectively, and connected them together with DR repeat
sequence to form crRNA precursor, and cloned into the expression plasmid
(Fig.3A). The results showed that Cas13b with three crRNAs produced by
one precursor expression vector were more efficient than that with the
same crRNAs from three independent vectors (Fig.3B-3E). However, for
Cas13d, there was no difference between the two strategies, even the
opposite result was observed (Fig.3F-3I).
The result above suggested that Cas13b might be more capable of
processing crRNA precursor than Cas13d.
Application of Cas13b and Cas13d in endogenous mRNA knock-down
To test the knock-down efficiency of Cas13 system to endogenous mRNAs,
crRNAs for Zeb1 were used, both Cas13b and Cas13d could
significantly reduce the expression of Zeb1 (Fig.4A, 4B). In
order to further study the positional effect of crRNAs, we selected 20
crRNAs for each Zeb1 and Dnmt3a mRNA to test the
knock-down efficiency. The results were generally consistent with the
above findings. Namely, crRNAs located in the middle of mRNA had higher
efficiency on average (Fig.4C, 4D, Fig.S2A, S2B).
The effect of crRNA length on the knock-down efficiency of Cas13b and
Cas13d was also investigated using Zeb1 and Dnmt3a as
targets. The results were in agreement with the above. Cas13b exhibited
high compatibility for crRNAs with a wide range of length, while Cas13d
required a relatively long crRNA (Fig.4E, 4F, Fig.S2C, S2D).
In order to know whether the precursor formed by concatenation of
multiple crRNAs could affect the knock-down efficiency of endogenous
genes, three crRNAs of Dnmt3a mRNA were concatenated with DR
repeats into the precursor sequence (Fig.4G). Neither Cas13b nor Cas13d
showed different efficiency compared to the mixed individual mature
crRNAs (Fig.4H). Moreover, to test whether precursors composed of crRNAs
targeting different genes could knock-down the genes simultaneously,
crRNA precursors targeting Dnmt3a , PbxipI and Zeb1mRNA were designed (Fig.4I). The results showed that for the three
genes, crRNA precursors had the same inhibitory efficiency as a mixture
of each mature crRNAs (Fig.4J-4L).
Off target effects of Cas13b and Cas13d
To investigate the off-target effects of Cas13b and Cas13d, RNA-seq was
performed. HEK293T cells were transfected with luciferase, crRNA and
Cas13b/Cas13d expression plasmids. The same transfection system but
without crRNA was served as the control groups. Both 20 nt and 30 nt
crRNAs against luciferase RNA were combined with Cas13b and Cas13d
respectively. The transfected cell samples were subjected to luciferase
assay to confirm the knock-down efficiency of Cas13 system. RNA-seq
results showed that the combination of Cas13d with 20nt crRNA had the
least differential genes, and there was no significant difference among
the other three groups (Fig. 5A-5D). The proportion of genes in
different fold change clusters to the total differential genes was
approximately the same in each group except the group of Cas13d with 20
nt crRNA (Fig.5F-5G). It is worth noting that 20nt crRNA is not optimal
for Cas13d, so the fact that the lower number of differential genes in
this group may be related to the insufficient knock-down of the system
(Fig. 5E).
The overlap of differential genes between groups were very small
(Fig.5H-5L). Go analysis showed that the differential genes were not
enriched in any certain pathways but dispersed in different pathways
(figures 5J-5M). These results indicated that most of the differential
genes were from random changes which might be caused by the off-target
effect of Cas13b or Cas13d.
Application of Cas13b and Cas13d in vivo
Further,we tried to explore the RNA knock-down ability of Cas13 system
in vivo. The DNA fragment expressing Cas13b and a crRNA against
luciferase were knocked into the Rosa26 locus of mouse genome.
The obtained transgenic mice (designated as Cas13bKI) were crossed with
mouse strain expressing luciferase (named as LucKI) from an expression
cassette inserted in the Rosa26 site of mice to obtain the double
transgenic mice (dTg) (Fig.6A). There was an obvious decrease in the RNA
expression levels (Fig.6B). And in vivo optical imaging experiment
showed that the luciferase expression indicated by luminance in dTg was
significantly decreased compared to LucKI mice no matter from the belly
or the back of animals, and in both male and female mice (Fig.6C, 6D).
A second transgenic mouse systemically expressing Cas13b or Cas13d and a
gRNA against mCherry was also constructed by knock-in the expression
vector into Rosa26 site. The mCherry expressing plasmid was
delivered to the liver of the transgenic mice by hydrodynamic injection
(Fig.6E). The results showed that compared to the control mice, both
protein and RNA expression of mCherry was significantly reduced in
transgenic mice at 72 hours after injection (Fig. 6F-6I).
Besides, we tested whether Cas13 RNA editing system could be used as
treatment for acute hepatic failure. It is reported that increased
expression of NF-kB and TNFa aggravates disease
progression in acute hepatic failure[35, 36]. Here, the plasmid
expressing Cas13b (or Cas13d) and a gRNA against NF-kB (orTNFa ) was injected via tail vein into mice by hydrodynamic
injection. At 48 hours after injection, the acute hepatic failure model
was induced by LPS/D-GalN administration (Fig.6J). The results showed
that mice with NF-kB or TNFa knock-down had a higher
survival rate. None of the mice with NF-kB knock-down by Cas13b
system died; Only one mouse died in Cas13b-TNFa group, Cas13d-NF-kB
group and Cas13d-TNFa group respectively. However, half of mice died in
the non-treatment group. Hematoxylin eosin staining showed that the
liver injury in the non-treatment group was more serious in terms of
pathological indicators such as interstitial hyperemia, hepatocyte
separation, liver histolysis and diffuse hepatocyte necrosis (Fig.6K).
Meanwhile, the RNA expression levels of NF-kB and TNFa in
knock-down groups were much lower compared to the non-treatment group
(Fig.6L-6M).