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\begin{document}

\title{The effects of length and sequence of gRNA on Cas13b and Cas13d activity
in vitro and in vivo}



\author[1]{Yuhui Liu}%
\author[1]{Jian Fei}%
\author[1]{Ping Jing}%
\author[1]{Yi Zhou}%
\author[1]{Jingyu Zhang}%
\author[1]{Jiahao Shi}%
\author[1]{Mengjie Zhang}%
\author[1]{Hua Yang}%
\affil[1]{Tongji University}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Cas13 are the only CRISPR/Cas systems found so far, which target RNA
strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves
RNA by the crRNA guidance. However, the effect of the characteristics of
the spacer sequences, such as the length and sequence preference, on the
activity of Cas13b and Cas13d remains unclear. Our study shows that
neither Cas13b nor Cas13d has a particular preference for the sequence
composition of gRNA, including the sequence of crRNA and its flanking
sites on target RNA. However, the crRNA, complementary to the middle
part of the target RNA, seems to show higher cleavage efficiency for
both Cas13b and Cas13d. As for the length of crRNAs, the most
appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15
nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30
nt crRNA can achieve good effect. Both Cas13b and Cas13d show the
ability to process precursor crRNAs. Our study suggests that Cas13b may
have a stronger precursor processing ability than Cas13d. There are few
in vivo studies on the application of Cas13b or Cas13d in mammals. With
the methods of transgenic mice and hydrodynamic injection via tail vein,
our study showed that both of them had high knock-down efficiency
against target RNA in vivo. These results indicate that Cas13b and
Cas13d have great potential for in vivo RNA operation and disease
treatment without damaging genomic DNA.%
\end{abstract}%



\sloppy


\textbf{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.

\textbf{Materials and Methods}

\textbf{Cell lines, culture, and transfection}

HEK293T cells were cultured in complete DMEM high glucose medium
(Hyclone) supplemented with 10\% heat-inactivated fetal bovine serum and
maintained in a humidified atmosphere of 5\% CO\textsubscript{2} at 37
\selectlanguage{ngerman}°C. Cells were transfected with plasmid by Lipofectamine\selectlanguage{english} 3000
Transfection Reagent (Thermo Fisher, USA) or Lipo8000\selectlanguage{english} Transfection
Reagent (Beyotime, China).

\textbf{Construction of Cas13b(d), gRNA expression plasmids}

The origin plasmids TC775, TC762, TC918, TC933 were purchased from
addgene (\emph{https://www.addgene.org}). For Cas13b gRNA expression
plasmid construction, SphI and SapI were used for digestion of TC775
(PCMV::Cas13b-P2A-GFP), the resulted large fragment was recovered by
electrophoresis. U6 promoter and DR sequence of crRNA in TC762
(PU6::PspCas13bDR) were amplified by PCR (forward primer:
5'-GAAGACAATAGCAGGcatgcGACTGCAGAGGtccgacgcc-3', reverse primer: 5'-
AGTGAGCGAGGAAGCGGAAGAGCGATCCAAAAAAAgttgtaat-3') according to the
condition: 2 min at 98degC, 35 cycles of 10s at 98degC, 15s at 56degC, and 20s
at 72degC, and a final step at 72 degC for 10 min. The PCR product purified
using Universal DNA Purification Kit (Tiangen) was cloned into the large
fragment with In-Fusion(r) HD Cloning Kit (TARAKA) to obtain the plasmid
of PCMV::Cas13b-P2A-GFP-PU6::PspCas13bDR. For the construction of Cas13d
gRNA expression plasmid, TC918 (PCAG::Cas13d-P2A-GFP) was linearized
using NotI and cloned with PCR products from TC933 (PU6:: CasRfx crRNA).
The primer sequences of PCR were as follows: (forward primer: 5'-
GGCGGGCATGCGGCCtccgacgccgccatctctag-3', reverse primer:
GTAAGTTATGCGGCCGAGTCAGTGAGCGAGGAAGC-3'). The plasmid obtained was
PCAG::Cas13d-P2A-GFP-PU6::CasRfx-crRNA.

The guide RNAs (gRNAs) against luciferase were designed in three
batches. The first batch was based on CRISPR-Cas system targeting data
(\emph{http://chopchop.cbu.uib.no}). The second batch was based on
Primer3 (\emph{https://primer3.ut.ee}) to select sequences with high
specificity. The third batch of gRNAs were selected randomly. The DNA
fragments for coding selected gRNAs were synthesized and cloned into the
plasmids obtained above by BsmBI restrict enzyme (NEB). The resulted
plasmid was designed as Cas13b-gRNA or Cas13d-gRNA respectively. All
gRNA sequences were listed in Table. S1. All plasmids were prepared
using Endotoxin-free plasmid extraction kit (Tiangen, China).

\textbf{Dual-luciferase reporter analysis}

For luciferase report assays, HEK293T cells were seeded in 96-well plate
(1x10\textsuperscript{4} cells/well) 24 hours before plasmid
transfection. The Cas13b-gRNA or Cas13d-gRNA plasmids with
pmirGLO-REPORT\selectlanguage{english} vectors were co-transfected into cells using
lipofectamine 8000 (Beyotime), fresh medium was added 24 hours after
transfection. Cells were assayed 48 hours after transfection using
Dual-Luciferase Reporter Assay System (Beyotime). Briefly, the cells
were lysed with cell lysis buffer (80 \selectlanguage{greek}μ\selectlanguage{english}l/well) on ice for 30 min. 30 \selectlanguage{greek}μ\selectlanguage{english}l
of cell lysis was mixed with 30 \selectlanguage{greek}μ\selectlanguage{english}l luciferase substrate in the dark, and
the luciferase emission was read by a single-tube chemiluminescence
detector. The renilla luciferase substrate was added to the mixture and
the renilla luciferase luminescence was read again. Renilla luciferase
activity was used as an internal reference.

\textbf{Quantitative Real-time PCR}

For checking the expression level of targeted mRNA in HEK293T, total RNA
was extracted from transfected HEK293T cells using Trizol (Tiangen,
China) according to the protocol of the manufacturer. 1 \selectlanguage{greek}μ\selectlanguage{english}g of RNA was
used for cDNA synthesis by using PrimeScript RT reagent Kit with gDNA
Eraser (Perfect Real Time, TAKARA, Japan) or TIANScript IIRT Kits (KR107,
TIANGEN, China). Quantitative real-time PCR was performed using the TB
Green\selectlanguage{ngerman}® Premix Ex Taq\selectlanguage{english} II (Tli RNaseH Plus, TAKARA, Japan). The relative
expression levels of genes were calculated and quantified by the method
of \selectlanguage{greek}ΔΔ\selectlanguage{english}Ct.

\textbf{Immunohistochemical analysis}

Immunohistochemical (IHC) staining was performed as follows: paraffin
sections were successively placed in xylene (two times), 100\% ethanol
(two times), 95\% ethanol, 85\% ethanol, 75\% ethanol, 70\% ethanol, and
PBS (two times), for 5 minutes each for dewaxing. The sections were
incubated in 3\% hydrogen peroxide for 10 minutes, and washed twice with
PBS. Then, the sections were placed in antigen repair solution preheated
to 100  and heated for another 20 minutes. Sections were blocked with
block solution for 25 minutes and incubated with the primary antibody
(polyclonal anti-mcherry, 26765-1-AP, proteintech, China) overnight.
After washing with PBST and PBS, sections were incubated with secondary
antibody (HRP-labeled Goat Anti-Rabbit IgG(H+L), A0208, Beyotime, China)
for 1 hour, and washed with PBST and PBS. DAB chromo developer
(Beyotime, China) was prepared and added to the sections by drops. After
chromo development for about 3 minutes, the sections were rinsed with
PBS. Hematoxylin was applied for 1 minute, and then the sections were
rinsed with water. The slices were successively dehydrated in 70\%,
75\%, 80\%, 95\%, 100\% ethanol (two times), and xylene (two times) for
5 minutes each, and then placed in a ventilated place to dry. Finally,
the slices were sealed with neutral resins (Solarbio, China) and
preserved.

\textbf{Cell flow cytometry.}

For testing the efficiency of d2eGFP targeting, flow cytometry was used.
HEK293T cells were seeded at 1.8\selectlanguage{ngerman}×10\textsuperscript{5} per well in a
24-well plate 18-24 h before transfection, and 1 \selectlanguage{greek}μ\selectlanguage{english}g remodeled plasmid
PCAG::Cas13d-P2A-d2eGFP-PU6::CasRfx-crRNA was transfected by
lipofectamine 8000. FACS was performed at 48 hours post-transfection.
All transfection experiments were performed in a way of biological
triplicates.

\textbf{RNA-seq libraries and analysis.}

Total RNA was extracted and qualified and sent to Berry Genomics
(Beijing, China) for transcriptome sequencing. After sequencing, obtain
a binary file in fastqc.gz format, then using Fastqc (version
0.11.7,\emph{https://www.bioinformatics.babraham.ac.uk/projects/fastqc/})
software for raw data quality assessment. Trim-galore (version
0.6.6,\emph{https://www.bioinformatics.babraham.ac.uk/projects/trim\_galore/,
parameters: paired, --quality 25 --length 36, stringency 3}) software
was used to remove splices and low-quality reads to obtain clean reads
with quality scores greater than Q20. Then, the human genome HG38
(\emph{https://hgdownload.soe.ucsc.edu/downloads.html\#hg38sequence})
and its annotation file
(\emph{https://hgdownload.soe.ucsc.edu/downloads.html\#hg38annotations})
were downloaded from UCSC website (\emph{http://www.genome.ucsc.edu/}),
which was used to construct human genome index by HISAT2 software
(hisat2, version 2.2.1,\emph{http://daehwankimlab.github.io/hisat2/ ;
parameters: default parameters}) and then aligned to the reference
genome. Samtools software (version
1.9,\emph{https://sourceforge.net/projects/samtools/files/samtools/1.9/;
parameters : -b -S -h}) was used to transform Sam files into BAM files
and sort them. After sorting, The featureCounts program in the subread
package (version 2.0.1,\emph{http://subread.sourceforge.net/ ;
parameters: -t exon -g gene\_id,}) was used for gene level
quantification. The results were expressed by FPKM. After obtaining the
count matrix, the R package DEseq2 (version
1.24.0,\emph{http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html})
based on the negative binomial distribution was used for the difference
analysis.

The fold change (FC) thresholds for identifying DEGs were \textbar{}
Log2FC \textbar{} \textgreater{}1, and a quality score greater than Q20,
i.e., false discovery rate (FDR) less than 0.05.and visualized the
results by R package ggplot2 {[}34{]}.

\textbf{Generation of CRIPSR/Cas13 mice.}

All systemic CRISPR/Cas13 expression mice were generated using the
CRISPR/Cas9 technique in Shanghai Model Organisms Center, Inc (Shanghai,
China). Briefly,a mixture of gRNA targeting Rosa26, Cas9 mRNA and Cas13
donor plasmid with 3.0 kb homologous arm was microinjected into one-cell
stage mouse embryos from C57BL/6 mice. The injected embryos were then
implanted into pseudo-pregnant C57BL/6 mice to generate F0 mice. The F0
mice were genotyped using PCR. The heterozygote mice were bred at the
Shanghai Model Organisms Center prior to starting any experiments. The
breeding and feeding were conducted according to Institutional Animal
Care and Use Committees protocols.

\textbf{Plasmid hydrodynamic injection and acute hepatic failure model.}

100 \selectlanguage{greek}μ\selectlanguage{english}g plasmid in 2 ml PBS solution was injected into the tail vein of
mice within 8-12 seconds. 8-week-old C57BL/6 mice were intraperitoneally
injected with LPS/D-GalN at 10 \selectlanguage{greek}μ\selectlanguage{english}L/g body weight (D-GalN: 200 mg/kg body
weight. LPS: 10 \selectlanguage{greek}μ\selectlanguage{english}g/kg body weight) respectively. After injection, the
mice were kept in a specific pathogen free (SPF) room, and the survival
status of mice was recorded in 24 hours.

\textbf{Statistical analysis.}

The significance of differences between groups was analyzed by ANOVA or
student T test, and P \textless{} 0.05 was considered to be
statistically significant. Results are expressed as the mean \selectlanguage{ngerman}± SD from
at least three independent experiments.

\textbf{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 (\emph{http://chopchop.cbu.uib.no}). The second was based on
Primer3 (\emph{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\selectlanguage{english}' PFS of the
original 30 nt crRNA or a 3 \selectlanguage{english}' 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 \emph{Zeb1} were used, both Cas13b and Cas13d could
significantly reduce the expression of \emph{Zeb1} (Fig.4A, 4B). In
order to further study the positional effect of crRNAs, we selected 20
crRNAs for each Zeb\emph{1} and \emph{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 \emph{Zeb1} and \emph{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 \emph{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 \emph{Dnmt3a} , \emph{PbxipI} and
\emph{Zeb1}mRNA 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\selectlanguage{english},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 \emph{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 \emph{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 \emph{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 \emph{NF-kB} and \emph{TNFa} aggravates disease
progression in acute hepatic failure{[}35, 36{]}. Here, the plasmid
expressing Cas13b (or Cas13d) and a gRNA against \emph{NF-kB}
(or\emph{TNFa} ) 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 \emph{NF-kB} or \emph{TNFa} knock-down had a higher
survival rate. None of the mice with \emph{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 \emph{NF-kB} and \emph{TNFa} in
knock-down groups were much lower compared to the non-treatment group
(Fig.6L-6M).

\textbf{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 \emph{Zeb1} and \emph{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 \emph{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 \emph{NF-kB} or \emph{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.

\textbf{Acknowledgments}

We appreciate Ms. Shen Jiajuan for the laboratory management affairs.
The research was funded by the National Key R\&D Program of China
{[}2019YFA0905900{]} and the Science and Technology Commission of
Shanghai Municipality {[}19DZ2280500, 18DZ2290700, 18DZ2293500{]}.

\textbf{Competing interests}

All authors declare that they have no conflict of any interest.

\textbf{Ethics approval}

All transgenic mouse were purchased from Shanghai Model Organisms
Center, Inc (Shanghai, China). 6-8-week-old C57BL/6 wild type mice were
purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai,
China). All experiments with mice were performed strictly in accordance
with a protocol approved by the Administrative Panel on Laboratory
Animal Care of Tongji University, Shanghai.

\textbf{Data Availability Statement}

Data available on request from the authors.

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\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig.1 Effect of gRNA~sequence on RNA knock-down efficiency. (A) The
CRISPR/Cas13 RNA targeting system. (B) A gradient curve for the
efficiency of Cas13b and Cas13d. Luc/Rluc plasmids are in a constant
volume, abscissa is the amount of Cas13b and Cas 13d, ordinate
represents the relative cutting efficiency of Cas13b and Cas13d. (C, D)
Different efficiency of 33 gRNAs is showed on the histogram. The
abscissa is ordered according to the location of gRNA on the mRNA. The
sequences of each corresponding gRNA are identical in Cas13b and Cas13d
systems. The red columns represent the first batch of gRNAs (Among them,
gRNA 2, 16, 29 represent up, mid, down gRNAs in figure.2A,2B), blue
columns represent the second batch of gRNAs, yellow columns represent
the third batch of gRNAs. All results are representative of at least
three independent experiments. (E) The relationship between GC content
and Cas13 efficiency.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig.2 Effect of gRNA length on RNA knock-down efficiency. (A, B) The
results of 30 nt gRNA of Luciferase. Renilla luciferase activity is used
as a reference. Samples transfected with Luc/Rluc and Cas13 but without
gRNA are considered negative control. (C, D) The results of 20 nt gRNA
of Luciferase. (E) The scheme describes the strategy for choosing
different PFS invariant gRNA. (F, G) The efficiency of different length
of gRNA action with same 5'PFS (F) and 3'PFS (G). (H, I) The efficiency
of different length of gRNA action with same 5'PFS (H) and 3'PFS (I) of
another site Luc3.12 which Cas13b showed better efficiency than Cas13d
with 30 nt gRNA. All results are representative of at least three
independent experiments. The results are presented as mean \selectlanguage{ngerman}± SD. Data
are analyzed by one-way ANOVA. *P \textless{} 0.05, **P \textless{}
0.01, ***P \textless{} 0.001, ****P \textless{} 0.0001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Fig.3 gRNA precursor processing ability of Cas13b/d. (A) The scheme
showing Cas13 processes the concatenated gRNA precursors into mature
gRNAs. u,m,d are gRNAs at up, mid and downstream locations respectively.
The ``+'' between gRNA symbols indicates that it is a mixture of
corresponding gRNA monomers, otherwise it is the precursor of
corresponding gRNA. (B-E) Effects of gRNAs form on Cas13b. (F-I) Effects
of gRNAs form on Cas13d. The results are presented as mean \selectlanguage{ngerman}± SD. *P
\textless{} 0.05, **P \textless{} 0.01, ***P \textless{} 0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig.4 Application of Cas13b(d) in endogenous gene knock-down. (A)
Knock-down of \emph{Zeb1} RNA expression by CRISPR/Cas13b. (B)
Knock-down of \emph{Zeb1} RNA expression by CRISPR/Cas13d. (C-D)
Knock-down of \emph{Zeb1} RNA expression by CRISPR/Cas13 with gRNA at
different position of target RNA. (E-F) Knock-down of \emph{Zeb1} RNA
expression by CRISPR/Cas13 with different length of gRNA. (G) The scheme
shows that 3 gRNAs targeting \emph{Dnmt3a} are produced with three
independent gRNA with one plasmid in precursor manner. (H) Knock-down
of\emph{Dnmt3a} RNA expression by CRISPR/Cas13 with mixed plasmids of
gRNA or gRNAs precursor plasmid. (I) The scheme shows that gRNA 1-3
targeting\emph{Dnmt3a} , \emph{PBXIPI} , and \emph{Zeb1} are produced
with one precursor plasmid. (J-L) Knock-down of RNA expression of
\emph{Dnmt3a} ,\emph{PBXIPI} , and \emph{Zeb1} simultaneously by
CRISPR/Cas13 with mixed plasmids of gRNAs or precursor plasmid.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Fig.5 Off-target Effects of Cas13b and Cas13d. (A-D) Volcanic map of
gene fold change, b20 (A), b30 (B), d20 (C), d30 (D) vs NC. The results
were presented as mean \selectlanguage{ngerman}± SD. Data are analyzed by one-way ANOVA. ****P
\textless{} 0.0001. (E) Luciferase expression in 293T cells after a
48-hours transfection, renilla luciferase activity is used as a
reference. (F-G) The numbers of differential genes among the four
groups. Different colored boxes represent different level of fold
change, and each column represents the total number of differential
genes. (H-I) Venn diagram shows differential genes overlapped among the
four groups of samples. (J-M) Arrangement of GO pathways enrichment
score. BP: Biological Process; CC: Cellular Component. MF: Molecular
Function.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Fig.6 Application of Cas13b and Cas13d in vivo. (A) The scheme describes
the cross strategy of Cas13-Luciferase knock-down mice and luciferase
expressing transgenic mice. (B) Luciferase RNA expression of mouse
liver. The results are presented as mean \selectlanguage{ngerman}± SD. Data are analyzed by
unpaired Student's t-test *P \textless{} 0.05, **P \textless{} 0.01,
***P \textless{} 0.001, ****P \textless{} 0.0001. (C-D) Luciferase
images of male (B) and female (C) dTg (offspring of Cas13b-Luc gRNA KI
mating with Luciferase mouse) (n=5). (E) The scheme describes the
process of mcherry knock-down in mouse liver. (F-G) The mcherry RNA
expression in mouse liver. The results are presented as mean ± SD. Data
are analyzed by one-way ANOVA. ****P \textless{} 0.0001. (H)
Representative flow cytometry profiles and quantification of the
frequencies of mcherry positive cell population. (I) The results of
immunohistochemistry represent the expression of mcherry protein in
mouse liver (n=5). (J) The scheme describes the process of generation of
acute hepatic failure model and the gene knock-down strategy of
CRISPR/Cas13. (K) Hematoxylin-eosin staining~results of mouse liver at
24 hours after injecting D-GalN/LPS (n=5), living or dead indicated the
animal state at the sampling time point. (L-M) \emph{NF-kB}
and\emph{TNFa} RNA expression in mouse liver. The results are presented
as mean ± SD. Data are analyzed by one-way ANOVA. ****P \textless{}
0.0001.

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