Adenovirus vector-mediated delivery of the CRISPR-CasRx system enables gene knockdown with suppressed collateral activity

Yuki Osakada; Sota Kidawara; Eiko Sakai; Akira Asayama; Fuminori Sakurai; Kahori Shimizu; Hiroyuki Mizuguchi

doi:10.1016/j.omtn.2026.103057

. 2026 Aug 12;37(3):103057. doi: 10.1016/j.omtn.2026.103057

Adenovirus vector-mediated delivery of the CRISPR-CasRx system enables gene knockdown with suppressed collateral activity

Yuki Osakada ¹, Sota Kidawara ¹, Eiko Sakai ¹, Akira Asayama ¹, Fuminori Sakurai ^2,³, Kahori Shimizu ¹, Hiroyuki Mizuguchi ^1,^4,^5,^6,^∗

PMCID: PMC13560539 PMID: 42725063

Abstract

The CRISPR-Cas13 system is a programmable gene knockdown tool widely used in basic and clinical research. Meanwhile, RNA-targeting by Cas13 exhibits collateral activity, i.e., cleavage of non-target RNAs, raising serious concerns. Accumulating evidence suggests the collateral activity comes from high expression levels of the Cas13 system. Therefore, controlled expression of the Cas13 system using viral vectors could solve the problem. Adenovirus vectors are efficient gene delivery vehicles and commonly used for clinical therapy. Here, we generated adenovirus vectors carrying a CRISPR-Cas13 system using CasRx (Ad-CasRx). Ad-CasRx efficiently knocked down the on-target reporter with suppressed collateral activity in cell lines, while plasmid transfection-mediated delivery exhibited strong collateral activity. Moreover, Ad-CasRx efficiently knocked down Pcsk9 in cultured murine hepatic cells without suppressing abundant endogenous RNAs, suggesting that collateral activity was largely suppressed when targeting endogenous genes with Ad-CasRx. Finally, we targeted hepatic Pcsk9 in mice by intravenous administration of Ad-CasRx. Pcsk9 mRNA and serum total cholesterol levels were reduced without affecting the expression of major genes related to cholesterol metabolism and hepatic function. Serum ALT levels were not elevated. Overall, controlled delivery of Ad-CasRx enabled in vivo gene knockdown without evident side effects from collateral activity or Cas13 expression itself in liver.

Keywords: MT: RNA/DNA editing, CRISPR-Cas13 system, collateral activity, adenovirus vector, gene knock down tool, gene therapy

Graphical abstract

Osakada and colleagues report that adenovirus vector-mediated delivery of CasRx reduces target RNA expression while limiting detectable collateral effects compared with plasmid transfection. These results support Ad-CasRx as a promising delivery platform for safer RNA-targeting applications.

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, first identified as natural bacterial defense systems against phages and plasmids, are now used as powerful genome-editing tools for a wide variety of applications in cultured cells and living organisms.¹^,²^,³^,⁴ The CRISPR-Cas system is a simple two-component system consisting of a Cas nuclease and a guide RNA that recognizes the target DNA or RNA through complementary base pairing. Recently identified Cas13 systems target and cleave single-stranded RNA (ssRNA) using a CRISPR RNA (crRNA) composed of a direct repeat (DR) and an adjacent spacer sequence typically 20–30 nucleotides (nt) in length.⁵^,⁶^,⁷^,⁸ The Cas13 protein binds to the crRNA via the DR, while the spacer sequence binds to the target RNAs. Base pairing between the spacer sequence and target RNAs is highly stringent, such that two mismatches in the spacer sequence can markedly reduce or abolish Cas13 activity, resulting in precise target recognition. In contrast, conventional RNAi technologies use seed sequences only 2–8 nt in length for target recognition.⁶^,⁹^,¹⁰^,¹¹ Thus, the CRISPR-Cas13 system has the potential to provide a promising RNA-silencing technology. In fact, the CRISPR-Cas13 system has been widely used for in vivo knockdown experiments either as ribonucleoproteins or virus vectors.¹²^,¹³^,¹⁴^,¹⁵^,¹⁶ Several studies have reported that adeno-associated virus (AAV)-mediated delivery of CRISPR-Cas13 successfully reduced target mRNAs in murine disease models, including mutant Htt mRNA in a Huntington disease model; Ataxin-2 in the brain and spinal cord of a TDP-43 proteinopathy model; and Vegfa mRNA in the retina of an age-related macular degeneration model, with accompanying improvements in pathological features.¹³^,¹⁴^,¹⁵^,¹⁷

Because CRISPR-Cas13 systems provide bacterial immunity against phages and plasmids by sensing invader transcripts and then nonspecifically destroying RNAs to arrest infected cells, they intrinsically have the potential to attack bystander RNAs when expressed in eukaryotic cells.⁵^,¹⁸^,¹⁹ Mechanistically, the Cas13 protein is converted to an activated form, exposing the active site of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) endoribonuclease on the surface when the crRNA binds its target, and a nonspecific RNA cleavage activity, termed collateral activity, is triggered.⁵^,¹⁸^,²⁰ In fact, recent studies found that collateral activity of Cas13 was evident when introduced into mammalian cells by plasmid transfection.²¹^,²² Several studies have reported that collateral activity correlates positively with target RNA expression levels, whether from artificially introduced reporter genes or endogenous genes.²¹^,²²^,²³ Cas13 protein and guide RNA expression levels have also been reported to modulate the extent of collateral activity.²²^,²⁴ Together, these findings suggest that restricting Cas13 expression may mitigate collateral activity. In this regard, viral vectors may be suitable for delivering Cas13 due to their ability to transduce cells more uniformly, often at relatively low copy number per cell. Consistent with this possibility, reduced collateral activity was observed when RfxCas13d was delivered at a limited copy number by lentiviral vectors.²⁵ However, in a CRISPR-Cas13-mediated knockdown experiment in adult mouse brain neurons, a single-copy Cas13 knock-in at the Rosa26 locus followed by AAV-mediated delivery of crRNAs caused mouse death attributable to collateral activity.²³ Thus, conditions that enable CRISPR-Cas13-mediated knockdown without detectable collateral activity are still under debate.

Adenovirus (Ad) vectors have been widely utilized for gene therapy due to their large capacity to carry various combinations of genes, their episomal nature, which prevents insertional mutagenesis in the host genome, and efficient transduction of either dividing or quiescent primary cells such as adult hepatocytes.²⁶^,²⁷ In addition, Ad vectors exhibit strong hepatotropism, with >90% of systemically administered particles accumulating in the liver, which enables preferential gene delivery to the liver.²⁶ Here, we report the generation of Ad vectors expressing CRISPR-Cas13 systems with two different orthologs, CasRx and PspCas13b, and investigated their knockdown efficiency and collateral activity in vitro and in vivo. To minimize Ad vector-associated liver injury, we employed an improved Ad-E4-122aT, which carries miR-122a-targeted sequences in the 3′ UTR of the E4 gene and suppresses the E4 gene expression in the liver via miR-122a-dependent regulation, thereby reducing hepatotoxicity compared with conventional Ad vectors. Our data suggest that CRISPR-CasRx-expressing Ad vectors successfully reduced target mRNA without showing marked collateral activity.

Results

Knockdown efficiency and collateral activity of CasRx, Cas13X.1, and PspCas13b delivered by plasmid transfection in human and mouse cell lines

Among over forty Cas13 orthologs, CasRx from Ruminococcus flavefaciens XPD3002, Cas13X.1 identified based on metagenomic data analysis, and PspCas13b from Prevotella sp. P5-125 were chosen for the initial in vitro study with the goal of developing Ad vectors because they have been utilized in knockdown studies using a lentiviral vector and an AAV but not an Ad vector.⁶^,⁷^,²⁸^,²⁹^,³⁰ In the previous studies using plasmid transfection-mediated delivery, all three of these Cas13 proteins efficiently reduced the gene expression from co-transfected reporter plasmids in HEK293T cells.²¹^,²⁹ In the same studies, CasRx reduced the gene expression from co-transfected collateral reporter plasmids, while PspCas13b showed slight collateral activity.²¹ Meanwhile, Cas13X.1 induced mild collateral activity for the stably transfected reporter gene expression when transiently transfected with an on-target reporter plasmid in HEK293T cells.²⁹

First, crRNAs targeting firefly luciferase (Fluc) mRNA were selected to examine their knockdown efficiency in HEK293 cells (Figure S1A). CasRx revealed efficient knockdown activities with all tested crRNAs, whereas PspCas13b showed the highest knockdown with crFluc4 and crFluc7 (Figure S1B). Thus, crFluc4 and crFluc7 were used in later experiments for CasRx and PspCas13b. Because Cas13X.1 showed only slight suppression with crFluc7, crRNAs with a longer spacer (30 nt), which showed efficient activity with Cas13X.1 in a previous study, were further tested; the results indicated that crFluc19, instead of crFluc7, was suitable for Cas13X.1 (Figure S1C).²⁹

Next, the knockdown efficiency and collateral activity of these Cas13 proteins delivered by plasmid transfection were evaluated in mouse and human cell lines. A dual-reporter system with a Fluc-expressing plasmid as a target reporter and the Gaussia luciferase (Gluc)-expressing plasmid as a non-target reporter was used for the analysis (Figures 1A and 1B). Plasmids expressing Cas13 and a crRNA targeting Fluc mRNA were co-transfected with the dual-reporter plasmids, and the knockdown efficiency and collateral activity were evaluated as reductions in Fluc and Gluc activity, respectively. Cas13 protein expression was confirmed in cells co-transfected with the Cas13-expressing plasmid and a non-targeting crRNA (Figure S2). CasRx efficiently knocked down Fluc expression in all cell lines at a level similar to shRNA (Figure S3), with a clear concomitant reduction in Gluc activity in A549, HepG2, SK-HEP-1, and AML12 cells, indicating robust collateral activity of CasRx in these cell lines (Figures 1C and 1D). However, no reduction in Gluc activity was observed in B16CAR cells, presumably due to the lower expression level of CasRx in B16CAR cells (Figure S2). Similarly, PspCas13b efficiently knocked down Fluc activity in all cell lines (Figure 1E). However, PspCas13b did not induce a significant reduction in Gluc activity, suggesting lower collateral activity of PspCas13b compared with CasRx (Figure 1F). In contrast to CasRx and PspCas13b, Cas13X.1 with crFluc4 revealed only a mild reduction in Fluc activity in A549 cells and no detectable reduction in other cell lines. Cas13X.1 with crFluc19 revealed a mild reduction in Fluc activity in most cell lines, but no reduction in SK-HEP-1 and B16CAR cells, indicating lower nuclease activity of Cas13X.1 compared with CasRx and PspCas13b in these cells (Figure 1G). Cas13X.1 reduced Gluc activity only when significant reductions in Fluc activity were observed (crFluc19 in A549, HepG2, and AML12 cells; crFluc4 in A549 cells), consistent with the notion that the collateral activity correlates with on-target nuclease activity (Figures 1G and 1H).²²

Knockdown efficiency and collateral activity of the various CRISPR-Cas13 systems introduced into cultured human and mouse cell lines by plasmid transfection

(A) A schematic showing the experimental design. Cultured human and mouse cells were co-transfected with reporter plasmids (pHMCMVL1 and pHMCMVG1) and Cas13-expressing plasmids carrying Fluc-targeting crRNAs (pHM5-U6-CMV-tetO2-CasRx-3xHA, pHM5-U6-PspDR-CMV-tetO2-PspCas13b-3xHA, or pHM5-U6-X.1DR-CMV-tetO2-Cas13X.1-3xHA). Knockdown efficiency and collateral activity were measured by the reduction in Fluc and Gluc activity, respectively. (B) A schematic diagram of the plasmid vectors used in this report. DR, direct repeat; tetO, tet operator; U6, U6 promoter; CMV, CMV promoter; HA, HA tag; pA, bovine growth hormone polyadenylation signal; Fluc, Firefly luciferase; Gluc, Gauusia luciferase; crFluc, Fluc-targeting crRNA. (C, E, and G) Fluc activities, relative to those with the non-targeting crRNA (crNT) in CasRx- (C), PspCas13b- (E), and Cas13X.1- (G)transfected in A549, HepG2, SK-HEP-1, AML12, and B16CAR cells, are shown (n = 4). (D, F, and H) Gluc activities, relative to those with the non-targeting crRNA (crNT) in CasRx- (D), PspCas13b- (F), and Cas13X.1- (H)transfected A549, HepG2, SK-HEP-1, AML12, and B16CAR cells, are shown (n = 4). Data are presented as means ± SD. Representative data from three independent experiments are shown. Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons post hoc test comparing to the control crNT.

Based on these observations, PspCas13b was a good candidate for generating Ad vectors with high on-target nuclease activity and low collateral activity. CasRx was also a candidate because it showed the highest knockdown activity among the three, and we hypothesized that Ad vector-mediated delivery might reduce collateral activity by restricting CasRx expression levels.²⁴

Generation of Cas13-expressing Ad vectors

To generate Ad vector plasmids, the same expression cassettes used in the plasmid transfection experiments, in which each Cas13 was regulated by a tet operator, were integrated into the E1 region of the Ad-E4-122aT Ad vector plasmid, in which hepatocyte-enriched microRNA miR122a target motifs were integrated downstream of the E4 gene to suppress the leaky expression of viral genes (Figure 2A).³¹ We previously found that Ad vectors carrying the CRISPR-Cas12a system were difficult to produce in the presence of Cas12a expression during vector propagation, suggesting that the bacterial-derived protein Cas13, like Cas12a, might inhibit the propagation of Ad vectors.³² Therefore, 293TetR10 cells stably expressing a tet repressor were newly established as a packaging cell line so that Cas13 expression would be suppressed during vector propagations. As a result, Ad vectors carrying CRISPR-Cas13 systems (Ad-CasRx and Ad-PspCas13b), as well as Ad vectors expressing two reporter genes (Ad-Fluc and Ad-Gluc), were successfully produced at reasonable titers (Table S2).

Knockdown efficiency and collateral activity of the various CRISPR-Cas13 systems introduced into cultured human and mouse cell lines by adenoviral transduction

(A) The schematic structure of Ad vectors used in this report. ITR, inverted terminal repeat; DR, direct repeat; tetO, tet operator; U6, U6 promoter; CMV, CMV promoter; HA, HA tag; pA, bovine growth hormone polyadenylation signal; Fluc, Firefly luciferase; Gluc, Gauusia luciferase; crFluc, Fluc-targeting crRNA. (B) A schematic showing the experimental design. Cells were co-transduced with Ad-Fluc and Ad-Gluc and with either Ad-CasRx/Ad-PspCas13b (crFluc) or Ad-shRNA. MOIs were as follows. SK-HEP-1: Ad-Fluc/Ad-Gluc, 3 each; Ad-CasRx/Ad-PspCas13b/Ad-shRNA, 100. AML12: 30 each; 300. B16CAR: 10 each, 300. Summary of Ad vector doses and expression levels of Cas and reporter genes for each cell line were shown in Table S3. (C and E) Fluc activities of Ad-CasRx (C) and Ad-PspCas13b (E), relative to those with the non-targeting crRNA (crNT)-expressing Ad vector in SK-HEP-1, AML12, B16CAR cells, are shown (n = 4). (D and F) Gluc activities of Ad-CasRx (D) and Ad-PspCas13b (F), relative to those with the non-targeting crRNA (crNT) in SK-HEP-1, AML12, B16CAR cells, are shown (n = 4). (G) Fluc activities of Ad-shRNA1 and -shRNA2, relative to those with Ad-null, are shown (n = 4). (H) Gluc activities of Ad-shRNA1 and Ad-shRNA2, relative to those with Ad-null in SK-HEP-1, AML12, and B16CAR cells, are shown (n = 4). Data are presented as means ± SD. Representative data from three independent experiments are shown. Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons post-hoc test comparing to the control non-targeting (crNT) (C–F) and Ad-null (G and H).

Knockdown efficiency and collateral activity of Ad-CasRx and Ad-PspCas13b in cells co-transduced with dual-reporter Ad vectors

Similarly to the plasmid transfection-mediated expression in Figure 1, the dual-reporter system was used to evaluate knockdown efficiency and collateral activity of Ad-CasRx and Ad-PspCas13b. Ad-Fluc and Ad-Gluc were used as an on-target reporter and a reporter for the collateral activity, respectively (Figures 2A and 2B). SK-HEP-1 cells, a human hepatocarcinoma-derived cell line, were used for the analysis because they are non-permissive for replication of E1-deleted Ad vectors.³³ In contrast, other human transformed cell lines, such as A549 and HepG2 cells, show leaky replication of Ad vectors, which could unintentionally increase expression of Cas13 and the reporter genes.³⁴^,³⁵ Murine transformed cell lines, AML12 and B16CAR cells, which are also non-permissive for Ad vector replication, were thus used here.³⁶ To mimic the condition in which both on-target knockdown and collateral activity come from targeting endogenous RNAs, cells are required to be transduced with the reporter Ad vectors at the minimum dose, preferentially a single copy per cell. In our previous study, approximately 50% of SK-HEP-1 cells were transduced with Ad vectors at an multiplicity of infection (MOI) of 3, and thus half the Gluc-expressing cells would theoretically co-express Fluc.³⁷ To equalize the Fluc activity, interpreted as the transduction efficiency, to that in SK-HEP-1 cells transduced with Ad vectors at an MOI of 3, the doses of reporter Ad vectors were set to MOIs of 30 and 10 for AML12 and B16CAR cells, respectively (Figure S4). Regarding effectors, Cas13 proteins from Ad-CasRx and Ad-PspCas13b showed comparable expression at MOIs of 100 and 300 in SK-HEP-1 and AML12 cells, respectively (Figures S5A and S5B). Despite the unexpectedly low expression levels of Cas13 proteins in B16CAR cells (Figures S5A and S5B), an MOI of 300 was set for B16CAR cells to exclude potential negative effects of overloading Ad vectors. Under the settled conditions, Ad-CasRx with on-targeting crRNAs caused an approximately 50% reduction in Fluc expression in SK-HEP-1 and AML12 cells and 70%–80% reduction in B16CAR cells, whereas only a slight reduction in Gluc expression was observed in B16CAR cells. This contrasted with the results obtained with plasmid transfection-mediated delivery (Figures 1D, 2C, and 2D). Similarly to Ad-CasRx, Ad-PspCas13b caused 50%–60% reductions in Fluc expression in SK-HEP-1 and AML12 and 93%–95% reductions in B16CAR cells (Figure 2E). Although Ad-PspCas13b did not induce significant reductions in Gluc expression in SK-HEP-1 and AML12 cells, it induced a 30%–50% reduction in B16CAR cells, indicating the collateral activity of Ad-PspCas13b (Figure 2F). Cells were also transduced with Ad vectors expressing Fluc-targeting-shRNA, Ad-shRNA1, and Ad-shRNA2, as a control, resulting in a significant reduction in Fluc expression in all the cell lines, with higher efficiency of shRNA2 than shRNA1 (Figure 2G). Neither Ad-shRNA1 nor Ad-shRNA2 induced a significant reduction in Gluc activity in SK-HEP-1 and B16CAR cells, as expected. However, both vectors induced mild reduction in Gluc activity in AML12 cells for unknown reasons, possibly from an off-target activity or hijacked RISC complexes as reported previously (Figure 2H).³⁸ Cell viability was assessed to examine the potential effects of Ad vector transduction, Cas13 expression, and collateral activity. Compared with Ad-null, the empty control vector, Ad-CasRx did not reduce cell viability in SK-HEP-1 or AML12 cells, nor did Ad-PspCas13b in AML12 cells (Figures S6A and S6B). In B16CAR cells, both Ad-CasRx and Ad-PspCas13b showed very small but significant reductions regardless of the targeting activity (Figure S6C). However, in SK-HEP-1 cells, Ad-PspCas13b with any of the crRNAs induced a mild but significant reduction in the cell viability, revealing the intrinsic toxic effect of PspCas13b protein expression (Figure S6A).³⁰ These observations suggested that the potential collateral activity of CasRx, which was evident in the plasmid transfection-mediated delivery, was reduced to an undetectable level while retaining the on-target knockdown activity in this assay context, when CasRx was delivered by Ad vectors. Because the abundance of on-target transcripts affects collateral activity and that of the non-target reporter might affect the sensitivity for detecting collateral activity, the mRNA copy numbers of reporter genes were next estimated in the plasmid transfection- and Ad vector-mediated delivery systems by quantitative reverse-transcription PCR (RT-qPCR). The Fluc transcript copy number was 10- to 100-fold higher in SK-HEP-1 and AML12 cells after plasmid transfection-mediated delivery than after Ad vector-mediated delivery (Figures S7A and S7B). Similarly, the Gluc transcript copy number was 5- to 10-fold higher in SK-HEP-1 and AML12 cells after plasmid transfection-mediated delivery (Figures S7A and S7B). Collateral activity was clearly observed with plasmid transfection-mediated delivery in these cell lines. Conversely, Fluc and Gluc transcript copy numbers in B16CAR cells were approximately 10- to 100-fold lower than those in SK-HEP-1 and AML12 cells after plasmid transfection-mediated delivery and were comparable to those after Ad vector-mediated delivery. Consistently, collateral activity was reduced to undetectable levels in B16CAR cells after both plasmid transfection-mediated and Ad vector-mediated delivery (Figures 1C and 1D; Figure S7C). Therefore, the differences in collateral activity between the two experimental systems were difficult to attribute to the CasRx delivery method.

Knockdown efficiency and collateral activity of Ad-CasRx and Ad-PspCas13b in the cells stably expressing the dual-reporter system

To exclude the effects from the different amount of reporter transcripts, we directly compared the two different delivery methods of CasRx using a reporter cell line next. An SK-HEP-1-derived cell line stably co-expressing Fluc and Gluc, named SK F/G, was generated and used for comparison of plasmid transfection-mediated and Ad vector-mediated delivery of CRISPR-Cas13 (Figure 3A). Unlike in the co-transduction system in Figure 2, the use of this reporter cell line ensured co-expression of the on-target reporter and the reporter for collateral activity in a single cell. When SK F/G cells were transduced with Ad-CasRx with crFluc4 or crFluc7, there was no reduction in Gluc expression relative to the non-targeting control, even under the condition in which on-target Fluc expression was largely suppressed (Figures 3B and 3C). Ad-CasRx with crFluc4 or crFluc7 reduced Fluc mRNA to ∼30% of the non-targeting control, whereas the mRNA levels of CasRx and Gluc were not significantly altered (Figures 3D and 3E). Next, to precisely evaluate the expression levels of reporter genes in plasmid-transfected cells, SK F/G cells were co-transfected with CasRx- and GFP-expressing plasmids, and GFP-positive cells were isolated by flow cytometry for quantification of the mRNA levels of CasRx, Fluc, and Gluc by RT-qPCR (Figure 3F). Enrichment of the GFP-positive cell population from 3% to 5%–∼70% was confirmed (Figure S9). In these isolated cells, CasRx with crFluc4 or crFluc7 reduced Fluc mRNA levels to ∼27% or ∼60% of the non-targeting control using crFluc4 primer set, respectively (Figure 3G). When measured using the crFluc7 primer set, CasRx with crFluc7 also tended to reduce Fluc mRNA levels to ∼30% of the non-targeting control, although it was not significant. In parallel, CasRx and Gluc mRNA levels decreased to 50% and 35%, respectively, indicating that substantial collateral activity was induced, as expected (Figure 3H). CasRx transcript levels in cells expressing non-targeting crRNA were approximately 7-fold higher after plasmid transfection than after Ad vector transduction (Figure S10). These results clearly indicate that the reduced collateral activity was attributed to the Ad vector-mediated controlled delivery of CasRx. Similarly, a modest but significant reduction in Fluc expression with no reduction in Gluc expression was observed in SK F/G cells when transduced with Ad-PspCas13b, as observed in plasmid transfection-mediated delivery in SK-HEP-1 cells (Figure 1; Figures S11A and S11B). SK F/G cells were also transduced with Ad vectors expressing Fluc-targeting shRNAs, Ad-shRNA1 and Ad-shRNA2, as controls, resulting in a significant reduction in Fluc expression to levels comparable to those achieved by Ad-CasRx, while no reduction in Gluc expression was observed (Figures S12A and S12B). Taken together, these results demonstrated that Ad-CasRx knocked down the on-target reporter gene expression more efficiently than Ad-PspCas13b and did not induce detectable suppression of non-targeted reporter gene expression. Considering the superior knockdown efficiency of Ad-CasRx along with the cellular toxicity of Ad-PspCas13b in SK-HEP-1 cells (Figure S6A), Ad-CasRx seemed preferable for further analysis.

Collateral activity of CRISPR-Cas13 system-expressing Ad vector in SK F/G cells

(A) A schematic showing the experimental design in (B–E). SK F/G cells, an SK-HEP-1-derived cell line stably expressing Fluc and Gluc, were transduced with Ad-CasRx at an MOI of 300. (B and C) Knockdown efficiency (B) and collateral activity (C) was evaluated by analyzing enzymatic activities of Fluc and Gluc, respectively. Luciferase activities in on-targeting crRNAs (crFluc4 and crFluc7) relative to those with the non-targeting crRNA (crNT)-expressing Ad vector are shown. Representative data from three independent experiments are shown (n = 4). Data are presented as means ± SD. (D and E) mRNA expression of Fluc (D) and CasRx and Gluc (E) was quantified by RT-qPCR. mRNA expression levels were normalized to *MT-CO2* and relative values to the control non-targeting crRNA (crNT)-expressing Ad vector are shown. Primer sets spanning the crFluc4 and crFluc7 target sites, crFluc4 primer (left) and crFluc7 primer (right), respectively, were used in (D). Schematic showing the locations of the crRNA and the primer set was shown in Figure S8. Results were presented as mean ± SD of three independent experiments. (F) A schematic showing the experimental design in (G and H). SK F/G cells were co-transfected with GFP-expressing plasmid (pHMCA5-GFP) and CasRx-expressing plasmid carrying crFluc4, crFluc7, or non-targeting crRNA (crNT). Empty plasmid, pHM5, was used as a control. The FACS profile for GFP-positive cells in each transfectant is shown in Figure S9. (G and H) mRNA expression of Fluc (G) and CasRx and Gluc (H) was quantified by RT-qPCR. mRNA expression levels were normalized to *MT-CO2* and relative values to the control non-targeting crRNA (crNT) are shown. Primer sets for the analysis for Fluc expression are as in (D). Representative data from three independent experiments are shown. Data are presented as means ± SD. (n = 3). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons post hoc test comparing to the control non-targeting (crNT).

Knockdown of Pcsk9 expression by Ad-CasRx

Next, the knockdown efficiency for an endogenous gene by Ad-CasRx was evaluated. PCSK9 is a protein secreted from hepatocytes that promotes LDL-cholesterol receptor degradation by stimulating internalization of the surface LDL receptor.³⁹^,⁴⁰ Accumulating evidence has demonstrated that functional inhibition or gene disruption of Pcsk9 in mouse livers reduces serum cholesterol levels.¹⁶^,⁴¹^,⁴² Thus, Pcsk9 was used to evaluate CRISPR-Cas13 system-mediated in vivo knockdown. To test Pcsk9 knockdown by Ad-CasRx, Ad vectors expressing CasRx with crRNAs against Pcsk9 mRNA (crPcsk9-1 and crPcsk9-2) were generated (Figure 4A). AML12 cells were transduced with these Ad-CasRx at an MOI of 300. Expression of CasRx in almost every cell was confirmed by immunostaining (Figure 4D). Pcsk9 mRNA expression levels 48 h post-transduction demonstrated that Ad-CasRx with crPcsk9-2 reduced Pcsk9 mRNA to ∼30% of the non-targeting control (Figure 4B). Because collateral activity has been reported to preferentially affect highly abundant transcripts, we selected abundant transcripts in AML12 cells based on RNA sequencing (RNA-seq) databases (GEO: GSE146053 and GSE165320) and analyzed their expression levels to assess potential collateral activity. mt-Co2 mRNA, which is encoded by the mitochondrial genome and thus spatially separated from cytoplasmic Cas13 proteins, was used as an internal control, as in a previous report.²² Consistent with minimal collateral activity, the expression of the most highly expressed genes (Eef1a1, Elf2, Fth1, S100a6, Vim, and Actb) was not reduced by Pcsk9-targeting Ad-CasRx compared with the non-targeting control, suggesting that collateral activity of CasRx on endogenous transcripts was reduced to undetectable level in this assay context when delivered by Ad vectors (Figure 4C).

Knockdown of *Pcsk9* by Ad vector-mediated expression of CasRx in AML12 cells

(A) The schematic structure of Ad vectors expressing crRNAs against *Pcsk9*. ITR, inverted terminal repeat; DR, direct repeat; tetO, tet operator; U6, U6 promoter; CMV, CMV promoter; HA, HA tag; pA, bovine growth hormone polyadenylation signal; crPcsk9-1 and -2, *Pcsk9*-targeting crRNA. (B and C) RT-qPCR analysis of *Pcsk9* (B) and non-targeting genes (C) mRNA expression in AML12 cells. *Actb*, actin beta; *Alb*, albumin; *Eef1a1*, eukaryotic translation elongation factor 1-alpha 1; *Elf2*, eukaryotic initiation factor 2; *Fth1*, ferritin heavy polypeptide 1; *S100a6*, S100 calcium-binding protein A6; *Vim*, vimentin. AML12 cells were transduced with Ad-CasRx at an MOI of 300 and analyzed 48 h after transduction. mRNA expressions normalized to *mt-Co2* are shown as relative values to the control non-targeting crRNA (crNT)-expressing Ad vector. Results were presented as mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons post hoc test comparing to the control non-targeting (crNT). (D) Immunofluorescence analysis of CasRx expression in AML12 cells. AML12 cells were transduced with Ad-null or crNT, crPcsk9-1 or crPcsk9-2-expressing Ad-CasRx at an MOI of 300 and stained with anti-HA antibody 48 h after transduction. Cell nuclei were stained with DAPI. Scale bars indicate 50 μm.

In vivo knockdown of Pcsk9 by Ad CasRx

Because collateral activity of CasRx targeting Pcsk9 was not evident in abundantly expressed genes in cultured cells, in vivo knockdown of Pcsk9 using Ad-CasRx was performed next. Mice were intravenously injected with Ad-CasRx carrying crPcsk9-2 at a dose of 1.5 × 10⁹ infectious units (IFU) (Figure 5A). Eleven days after injection, the liver Pcsk9 mRNA levels in mice injected with Pcsk9-targeting Ad-CasRx were significantly reduced to approximately one-half of the non-targeting control level (Figure 5B). The serum PCSK9 protein levels showed a decreasing trend to almost one-half of the control level, although it was not significant (Figure 5C). Consistent with PCSK9 protein depletion, total serum cholesterol was significantly reduced to ∼60% of the control level (Figure 5D). To examine any unintended effects which might be caused directly or indirectly by CasRx collateral activity—and that could account for the reduced serum cholesterol levels—we assessed hepatic expression of genes related to cholesterol biosynthesis: HMG-CoA reductase (Hmgcr), ATP citrate lyase (Acly), and farnesyl diphosphate farnesyl transferase 1 (Fdft1); cholesterol efflux: secretion associated Ras related GTPase 1B (Sar1b), homolog A, COPII coat complex component (Sec24a), surfeit gene 4 (Surf4), acetyl-coenzyme A acetyltransferase 2 (Acat2), angiopoietin-like 3 (Angptl3), apolipoprotein B (Apob), and microsomal triglyceride transfer protein (Mttp); HDL-mediated cholesterol efflux and uptake: ATP-binding cassette, sub-family A member 1 (Abca1), and scavenger receptor class B, member 1 (Scarb1).⁴³^,⁴⁴^,⁴⁵ Expression of these genes in Ad-CasRx-injected mouse livers was comparable to that in the control (Figure 5E). Genes related to the hepatic functions: albumin (Alb), and hepatic nuclear factor 4 (Hnf4a) as well as the upstream regulator of metabolic enzymes in hepatocytes, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), were also expressed at the comparable levels to those in the control in Ad-CasRx-injected mouse livers (Figure 5F). These results demonstrated that the hepatic functions, specifically the major hepatic cholesterol metabolic pathways, were not largely impaired, suggesting that the decreased serum cholesterol level was the direct effect of Pcsk9 knockdown. Serum alanine aminotransferase (ALT) levels in each group did not exceed the reported range of healthy mice and were not elevated in mice injected with Pcsk9-targeting Ad-CasRx compared with either PBS or non-targeting Ad-CasRx controls, further supporting the hepatic safety of Ad-CasRx under these conditions (Figure 5G).⁴⁶ Taking these results together, Ad-CasRx achieved in vivo knockdown of Pcsk9 and a reduction in serum cholesterol, without showing detectable collateral activity on non-target genes related to cholesterol metabolism or hepatotoxicity.

Knockdown of *Pcsk9* by Ad vector-mediated expression of CasRx in the mouse liver

(A) A schematic showing the experimental design. Five-week-old female C57BL/6J mice were intravenously injected with the *Pcsk9*-targeting crRNA-expressing Ad-CasRx at 1.5 × 10⁹ infectious units (IFU) per mouse via the tail vein. Blood and liver were harvested at 11 days post-injection (dpi). ITR, inverted terminal repeat; DR, direct repeat; tetO, tet operator; U6, U6 promoter; CMV, CMV promoter; HA, HA tag; pA, bovine growth hormone polyadenylation signal; crPcsk9-2, *Pcsk9*-targeting crRNA-2. (B) RT-qPCR analysis of *Pcsk9*. (C) Serum PCSK9 protein expressions at 11 dpi are shown. (D) The total serum cholesterol levels at 11 dpi are shown. (E) RT-qPCR analysis of mRNA expression of non-targeting genes in the liver. *Abca1*, ATP-binding cassette, sub-family A member 1; *Acat2*, acetyl-coenzyme A acetyltransferase 2; *Acly*, ATP citrate lyase; *Angptl3*, angiopoietin-like 3; *Apob*, apolipoprotein B; *Fdft1*, farnesyl diphosphate farnesyl transferase 1; *Hmgcr*, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; *Mttp*, microsomal triglyceride transfer protein; *Sar1b*, secretion associated Ras related GTPase 1B; *Scarb1*, scavenger receptor class B, member 1; *SEC24a*, homolog A, COPII coat complex component; *Surf4*, surfeit gene 4. (F) mRNA expression in the liver. *Alb*, albumin; *Gapdh*, glyceraldehyde-3-phosphate dehydrogenase; *Hnf4a*, hepatic nuclear factor 4. (B, E, and F) mRNA expressions normalized to *mt-Co2* are shown as relative values to the control non-targeting crRNA (crNT)-expressing Ad vector. (G) Serum ALT was quantified at 11 dpi. Results were presented as mean ± SD (n = 6). Statistical significance was determined using Student’s t test in (B–F) and by one-way ANOVA with Dunnett’s multiple comparisons post hoc test comparing to the control injection with PBS in (G).

Discussion

Previous plasmid-based in vitro studies have shown that the collateral activity of the CRISPR-Cas13 system depends on the expression level of the target transcript, and higher expression level of Cas13 proteins further enhances collateral activity.²²^,²⁴ These observations strongly suggested that delivering the CRISPR-Cas13 system to target cells at low copy number could minimize collateral activity while preserving precise on-target knockdown. Here, we successfully demonstrated that Ad vector-mediated delivery of the CRISPR-Cas13 system was fully effective for on-target gene knockdown with suppressed collateral activity either for non-target reporter or endogenous genes in cell cultures.

Starting with the three Cas13 orthologs with well-established knockdown ability, the most appropriate one was narrowed down using our dual-reporter system. Plasmid-mediated delivery of CasRx and PspCas13b achieved high levels of target-gene knockdown. However, Cas13X.1 failed to achieve consistent knockdown over the tested cell lines, in sharp contrast to the previous reports in HEK293T cells.²⁹ This discrepancy might simply reflect differences in transfection efficiency (HEK293 cells vs. other cells) or cell-type-dependent post-translational modifications, as reported for SpCas9, in which SUMOylation in the HNH nuclease domain modulates crRNA-directed DNA-binding activity and ubiquitination in the RuvC III domain regulates protein stability.⁴⁷ However, western blot analysis showed similar protein levels of Cas13X.1 and PspCas13b, suggesting that differences in expression or protein stability are unlikely to explain the impaired knockdown activity of Cas13X.1 (Figure S2). Alternatively, Cas13X.1 might require a high concentration of protein to exert its enzymatic activity, and thus Cas13X.1 showed knockdown activity only in cells competent for highly efficient transfection, such as HEK293 cells (Figure S1C). In addition, the crRNA for Cas13X.1 was likely not optimized, although 15 crRNAs targeting Fluc were tested in the present study. Further optimization of crRNA design might improve Cas13X.1-mediated knockdown in various cell lines. Consistent with the previous reports, plasmid transfection-mediated delivery of CasRx exhibited robust collateral activity in all cell lines, whereas PspCas13b did not.²¹ However, Ad-PspCas13b unexpectedly induced collateral activity in B16CAR cells with highly efficient target-gene knockdown (93%–95%) despite the low level of protein expression (Figure S5). Ad-PspCas13b also induced cytotoxicity in SK-HEP-1 cells independent of its target-gene knockdown activity (Figure S6A). In contrast, Ad-CasRx did not induce either detectable collateral activity or cytotoxicity in any of the cultured cell lines in this study. Notably, we demonstrated that CasRx copy numbers were much lower in cells receiving Ad vector-mediated delivery than in those receiving plasmid transfection-mediated delivery (Figure S10), supporting the notion that low-copy delivery of CasRx by an Ad vector reduced collateral activity. Hara et al. showed that a limited copy number of CasRx (also known as RfxCas13d) reduced collateral activity using lentiviral vector systems, further supporting our present observations.²⁵ Indeed, Ad-CasRx knocked down the endogenous Pcsk9 gene with no marked reduction in abundant endogenous transcripts in AML12 cells (Figures 4B and 4C). Taken together, these results show that controlled delivery by Ad vectors potentiates CRISPR-CasRx as a knockdown tool free from robust collateral activity and cytotoxicity.

Importantly, Ad-CasRx successfully reduced Pcsk9 expression to 50% in vivo, resulting in a decline in serum cholesterol levels to ∼60% in mice (Figures 5B and 5D). Similar mouse studies using siRNA demonstrated a 50%–70% reduction in hepatic Pcsk9 mRNA accompanied by up to ∼60% decreases in total serum cholesterol. Other studies showed that RNAi-based therapeutics targeting Pcsk9 reduced serum PCSK9 protein levels to 25%–50% and LDL-cholesterol to 50%–70% compared with baseline.⁴⁸^,⁴⁹^,⁵⁰ Therefore, the extent of Pcsk9 knockdown and reduction of total serum cholesterol achieved in our study is plausibly relevant to a therapeutic range. Notably, these reductions occurred in the absence of disordered expression in major cholesterol metabolism and hepatic function-related genes and elevation of ALT, highlighting Ad-CasRx as a promising in vivo RNA-targeting tool for therapeutic use.

Contrary to our observations that Ad-CasRx showed no obvious hepatic toxicity and disturbance of gene expressions, Li et al. reported neuronal toxicity of CasRx in in vivo.²³ Specifically, AAV-mediated delivery of crRNAs targeting endogenous genes induced lethality within 4 weeks in neuron-specific, conditionally expressing CasRx knock-in mice. This discrepancy with our observations could be attributed to the difference in experimental period (4 weeks vs. 11 days) or in a tissue-dependent threshold of tolerance for collateral activity (brain neurons vs. liver). From this point of view, it is noteworthy that Cas13 orthologs showed unexpectedly variable knockdown and collateral activities depending on the cell line and delivery method, as we observed. CasRx-associated toxicity or collateral activity may become apparent after prolonged expression or in tissues with lower tolerance thresholds. Therefore, caution is warranted when directly applying our Ad-CasRx to tissues other than liver or when using different administration protocols. More detailed studies will be required to define the expression thresholds and durations associated with collateral activity and toxicity in various tissues. Taken together, our findings and those of others suggest that in vivo application of CasRx requires thorough assessments to avoid toxic effects in the target tissue type.

Although our controlled delivery of CasRx by Ad vector did not induce liver damage, the residual collateral activity would be a point of concern when using this delivery method in other tissues. Recently, a high-fidelity Cas13d variant (hfCas13d) was developed that maintains on-target activity while showing substantially reduced collateral activity compared with wild-type CasRx.⁵¹ In in vivo experiments, this variant was shown not to induce appreciable collateral activity either in transgenic mice or in somatic cells targeted by AAV-based delivery.⁵¹^,⁵² hfCas13d successfully restored transcriptional abnormalities in an amyotrophic lateral sclerosis-frontotemporal dementia (ALS/FTD) mouse model through precise cleavage of the mutant C9ORF72 transcript, supporting its potential for therapeutic applications.⁵² In addition, a negative autoregulation system for CasRx itself, gRNA excision for negative-autoregulatory optimization (GENO), was developed to minimize the collateral activity by controlling Cas protein expression.²⁴ Combined with these new technologies, Ad-CasRx would be a more secure therapeutic knockdown tool applicable for a wide range of tissues.

For viral vector-mediated delivery of an improved CRISPR-Cas13 system, Ad vectors are preferable to AAV because of the larger packaging capacity (≤8 kb), allowing, for example, Cas13 to self-mature crRNAs from a crRNA array and thus target multiple RNAs. Theoretically, Ad genes are not expected to be expressed following transduction with a replication-incompetent Ad vector; however, leaky expression can occur, resulting in the induction of cellular immunity against Ad proteins as well as Ad protein-mediated hepatotoxicity. To mitigate these liabilities, we used Ad-E4-122aT, which can reduce Ad vector-associated hepatotoxicity by suppressing E4 gene expression in the liver via miR-122a-mediated regulation, thereby attenuating both adaptive and innate immune responses.³¹^,⁵³ Alternatively, helper-dependent (gutless) Ad vectors that lack all viral coding regions have been reported to reduce tissue inflammation.⁵⁴^,⁵⁵ Meanwhile, the CRISPR-Cas13 system is preferable to shRNA-mediated knockdown for Ad vector delivery because Ad vector-encoded VA-RNA competitively inhibits shRNA biogenesis, thereby reducing knockdown efficiency.⁵⁶ Therefore, CRISPR-Cas13-loaded Ad vectors represent a suitable platform for in vivo RNA knockdown.

Finally, several limitations in this study should be acknowledged. First, the threshold doses for collateral activity of Ad-CasRx remain to be determined both in vitro and in vivo. Latent collateral activity at the doses without apparent phenotypic changes also needs to be addressed by comprehensive analysis of RNA expression levels in cells and liver tissues. Even though our findings demonstrated that Ad-CasRx is a potentially useful platform for RNA knockdown with suppressed collateral activity in our systems, its safety and efficacy are likely context-dependent and may vary according to vector dose, target tissue, duration of expression, and delivery strategy. Therefore, further careful evaluations will be necessary to define the conditions under which Ad-CasRx can achieve efficient target knockdown while minimizing collateral activity and toxicity, particularly when applied in vivo.

Materials and methods

Cells and reagents

HEK293 cells (a human transformed embryonic kidney cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (043-30085, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine (Nacalai Tesque, Kyoto, Japan), 100 μg/mL streptomycin (Nacalai Tesque), and 100 U/mL penicillin (Nacalai Tesque). A549 (a human lung adenocarcinoma cell line), HepG2 (a human liver cancer cell line), and SK-HEP-1 (a human liver adenocarcinoma-derived endothelial cell line) cells were cultured in DMEM supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin. B16CAR cells (a murine melanoma cell line expressing coxsackievirus and adenovirus receptor) were previously generated and were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin.⁵⁷ AML12 cells (a mouse hepatocyte cell line) were cultured in DMEM/F-12 (Thermo Fisher Scientific, Waltham, MA) with 10% FBS, 1% (vol/vol) Insulin-Transferrin-Selenium solution (#41400045, Thermo Fisher Scientific), 40 ng/mL dexamethasone (FUJIFILM Wako Pure Chemical Corporation), 100 μg/mL streptomycin, and 100 U/mL penicillin. HEK293, A549 and AML12 cells were obtained from ATCC (ATCC, Manassas, VA). HepG2 cells were obtained from JCRB Cell Bank (JCRB Cell Bank, Osaka, Japan).

Plasmids

pT3TS-RfxCas13d-HA (#141320), pC0046-EF1a-PspCas13b-NES-HIV (#141320), and CMV-Cas13X.1-SV40pA_U6-BbsI-DR_CMV-mCherry-BGHpA (#171379) were obtained from Addgene.⁶^,²⁹^,⁵⁸ The backbone vector for loading the Cas13 system, pHM5-U6-CMV-tetO2-3xHA, was generated by integrating the chemically synthesized DNA fragment (Figure S13) between the I-CeuI and KpnI sites of pHM5.⁵⁹ pHM5-U6-CMV-tetO2-CasRx-3xHA was generated by cloning the DNA fragment for the CasRx coding sequence from pT3TS-RfxCas13d-HA into pHM5-U6-CMV-tetO2-3xHA by BglII and NotI digestion. The DRs for PspCas13b and Cas13X.1 were amplified by PCR from pC0046-EF1a-PspCas13b-NES-HIV and CMV-Cas13X.1-SV40pA_U6-BbsI-DR_CMV-mCherry-BGHpA by using primers 1 and 2 and primers 3 and 4 in Table S4, respectively, and integrated into pHM5-U6-CMV-tetO-3xHA between the MluI and MfeI sites to generate pHM5-U6-PspDR-CMV-tetO-3xHA and pHM5-U6-X.1.DR-CMV-tetO-3xHA. The coding regions of PspCas13b and Cas13X.1 were amplified by PCR from pC0046-EF1a-PspCas13b-NES-HIV and CMV-Cas13X.1-SV40pA_U6-BbsI-DR_CMV-mCherry-BGHpA by using primers 3 and 6 and primers 7 and 8 in Table S4, respectively, and integrated into pHM5-U6-PspDR-CMV-tetO-3xHA and pHM5-U6-X.1DR-CMV-tetO-3xHA between the BglII and NotI sites to generate pHM5-U6-PspDR-CMV-tetO2-PspCas13b-3xHA and pHM5-U6-X.1DR-CMV-tetO2-Cas13X.1-3xHA. The DNA fragment for each crRNA was cloned into the BbsI site of pHM5-U6-CMV-tetO2-CasRx-3xHA, pHM5-U6-PspDR-CMV-tetO2-PspCas13b-3xHA, or pHM5-U6-X.1DR-CMV-tetO2-Cas13X.1-3xHA. The oligonucleotides for crRNA sequences are listed in Table S1. The plasmid expressing Fluc, pHMCMVL1, was previously described.⁵⁹ The plasmid expressing Gluc, pHMCMVG1, was generated by cloning the PCR fragment amplified from pGluc-basic2 using primers 9 and 10 in Table S4 between the XbaI and NotI sites of pHMCMV6.⁵⁹ pHMU6-shRNA1 was previously described.⁶⁰ pHMU6-shRNA2 was generated by inserting the oligonucleotides encoding the shRNA for Fluc into pHMU6 at the BbsI site. The oligonucleotides for shRNA sequences are listed in Table S1. For establishing 293tetR10 cells, pENTR-Donor-EF1α-puroR was chemically synthesized and integrated with the coding region of the tetracycline repressor (tetR) gene, generating pENTR-LAD6-tetR-2A-Puro. A schematic diagram of pENTR-LAD6-tetR-2A-Puro is shown in Figure S14. The plasmid expressing GFP, pHMCA5-GFP, was previously described.⁶¹

Immunoblotting

Cells were seeded at a density of 1.0 × 10⁵ cells per well in 24-well plates. Twenty-four hours after seeding, the cells were transfected with 100 ng of pHM5-U6-CMV-tetO2-CasRx-3xHA, pHM5-U6-CMV-tetO2-PspCas13b-3xHA, or pHM5-U6-CMV-tetO2-Cas13X.1-3xHA expressing non-targeting crRNA using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. For the analysis using Ad vectors, cells were seeded at a density of 1.0 × 10⁵ cells per well in 24-well plates and transduced with Ad-null, Ad-CasRx, or Ad-PspCas13b at the indicated MOIs. Cells were lysed with RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma-Aldrich) 48 h after transfection and transduction. Six μg of proteins were separated by SDS-PAGE using a 10% polyacrylamide gel and transferred to a Immobilon-P PVDF membrane (Merck Millipore, Darmstadt, Germany). Following transfer, membranes were stained with Ponceau-S Staining Solution (Beacle, Kyoto, Japan) according to manufacturer’s protocols. Total protein loading was assessed by Ponceau-S staining and quantified as total lane density in ImageJ (NIH). Membranes were blocked with 5% skim milk in TBS containing 0.1% Tween 20 (TBS-T), incubated with primary antibodies overnight at 4°C, and then with HRP-conjugated secondary antibodies for 1 h at room temperature. Anti-HA antibody (1:3,000 in 1% BSA/TBS-T; #MMS-101P, BioLegend, San Diego, CA), anti-GAPDH (1:5,000 in 5% skim milk/TBS-T; #2275-PC-1, Trevigen, Gaithersburg, MD), and HRP-labeled goat anti-rabbit and horse anti-mouse antibody (1:5,000 in 5% skim milk/TBS-T; #7074 and #7076, Cell Signaling Technology) were used. Bands were visualized using Chemi-Lumi One Super (Nacalai Tesque) and images were captured with LAS3000 (FUJIFILM, Tokyo, Japan). Band intensities were quantified using ImageJ.

Packaging cells for Ad vector production

A packaging cell line 293TetR10 was generated as follows: HEK293 cells were transfected with pENTR-LAD6-tetR-2A-Puro linearized by NotI-digestion. Forty-eight hours after transfection using Lipofectamine 3000, cells were cultured in the presence of 10 μg/mL of puromycin (Thermo Fisher Scientific) for 2 days and 1.5 μg/mL of puromycin afterward, to isolate colonies of the stably TetR-expressing clonal cells.

Generation of SK-HEP-1 cells stably expressing Fluc and Gluc

SK-HEP-1 cells were transfected with pENTR-Donor-EF1α-puroR linearized by AsiSI-digestion, and pHMCMVL1 and pHMCMVG1 linearized by BbsI-digestion using Lipofectamine 3000. Forty-eight hours after transfection, cells were cultured in the presence of 2 μg/mL of puromycin for 2 days and reseeded to isolate colonies for cloning. Each clone was assessed for Fluc and Gluc expression using a Bright-Glo Luciferase Assay System (Promega, Madison, WI) and a Pierce Gaussia Luciferase Glow Assay Kit (Thermo Fisher Scientific), respectively.

Virus

Ad-null, an empty Ad vector, and Ad-Fluc (Ad-L2), an Fluc-expressing Ad vector, were previously described.⁶² The Ad vector plasmids for expression of Gluc (Ad-Gluc), shRNA (Ad-shRNA1 and Ad-shRNA2), CasRx (Ad-CasRx), and PspCas13b (Ad-PspCas13b) were prepared by an improved in vitro ligation method.⁶³ Briefly, the fragments of expression cassettes from pHMCMVG1, pHMU6-shRNA1, pHMU6-shRNA2, pHM5-U6-CMV-tetO2-CasRx-3xHA, and pHM5-U6-CMV-tetO2-PspCas13b-3xHA were individually integrated into the I-CeuI and PI-SceI sites of pAd-E4-122aT where the hepatocyte-enriched microRNA miR122a target motifs were integrated downstream of the E4 gene to suppress the leaky expression of viral genes.³¹ According to the standard protocol, Ad vector plasmids were transfected into 293TetR10 cells using Lipofectamine 2000 (Thermo Fisher Scientific) and Ad vectors were purified from cell lysates by two rounds of cesium-chloride-gradient ultracentrifugation. The infectious units (IFU) were determined using an Adeno-X Rapid Titer Kit (TAKARA BIO, Otsu, Japan). For transducing cells, cells suspended in the culture media were mixed with Ad vectors at the indicated MOI and were subsequently seeded into separated wells.

Mouse experiments

Mice were housed under a 12-h light/dark cycle with ad libitum access to CRF-1 (Oriental Yeast, Tokyo, Japan) and water. Five-week-old female C57BL/6J mice (Japan SLC, Hamamatsu, Japan) were intravenously injected via the tail vein with Ad-null or Ad-CasRx carrying a non-targeting or a Pcsk9-targeting crRNA (crPcsk9-2; Table S1, no. 17) at a dose of 1.5 × 10⁹ IFU per mouse. Mice were euthanized on day 11, and blood and livers were harvested for further analysis. All animal experimental procedures used in this study were performed in accordance with the institutional guidelines for animal experiments at The University of Osaka. All animal experimental procedures used in this study were approved by the Animal Experiment Committee of The University of Osaka (approval IDs doyaku R03–1 and R06-3) and performed in accordance with the institutional guidelines for animal experiments at The University of Osaka.

Serum analysis

After incubation of blood samples overnight at 4°C, serum was separated by centrifugation and frozen at −80°C until analyses. Serum levels of PCSK9 were determined using the mouse PCSK9 ELISA kit (#KE10050, Proteintech, Rosemont, IL) according to the manufacturer’s protocol. ALT and total cholesterol were measured by using LabAssay ALT (GPT) and LabAssay Cholesterol (#293–97501 and #291–93601, FUJIFILM) according to manufacturer’s protocols.

Real-time RT-PCR

Total RNA was isolated from mouse liver tissue samples or cultured cell lines using ISOGEN (NIPPON GENE, Tokyo, Japan). cDNA was synthesized using 500 ng of total RNA with a Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific). Real-time reverse-transcription PCR (real-time RT-PCR) was performed with SYBR Green PCR Master Mix (Thermo Fisher Scientific) using a StepOnePlus real-time PCR system (Applied Biosystems). The relative quantitation of target mRNA expressions was performed using the ΔΔCT method, using SYBR Green Master Mix (Applied Biosystems, A25741). Relative transcript abundance was normalized to mt-Co2. The sequences of the specific primers are shown in Table S5. For RT-qPCR quantification of CasRx, Fluc, and Gluc mRNA, primer sets No. 43 and 44, No. 45 and 46, and No. 47 and 48 listed in Table S5 were used, respectively. Absolute RT-qPCR of CasRx, Fluc, and Gluc mRNA was performed using serial dilutions of pHM5-U6-CMV-tetO2-CasRx-3xHA, pHMCMVL1, and pHMCMVG1 as templates for standard curves, respectively.

In vitro luciferase assay

Cells were seeded on a 96-well plate at 1.0 × 10⁴ cells/well. Twenty-four hours after seeding, the cells were transfected with 25 ng each of pHMCMVL1 and pHMCMVG1 and 50 ng of pHMU6-shRNA1, pHMU6-shRNA2, pHM5-U6-CMV-tetO2-CasRx-3xHA, pHM5-U6-CMV-tetO2-PspCas13b-3xHA, or pHM5-U6-CMV-tetO2-Cas13X.1-3xHA using Lipofectamine 3000. For the analysis using Ad vectors, the cells were transduced with Ad-Fluc and Ad-Gluc along with Ad-CasRx, Ad-PspCas13b, Ad-shRNA1, or Ad-shRNA2 at the indicated MOIs. To evaluate the KD efficiency, Fluc activity was measured 48 h post-transfection using the Bright-Glo Luciferase Assay System. To evaluate collateral activity, cell supernatants were collected and Gluc activity was analyzed with the Pierce Gaussia Luciferase Glow Assay Kit.

Measurement of cell viability

Cells were seeded on a 96-well plate at 1.0 × 10⁴ cells/well and transduced with Ad-null, Ad-shRNA1, Ad-shRNA2, Ad-CasRx, or Ad-PspCas13b at the indicated MOIs. Forty-eight h post-transduction, cell viabilities were determined using Cell counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol.

Fluorescence-activated cell sorting

SK F/G cells were seeded on 60 mm dish at 1.0 × 10⁶ cells/dish. Twenty-four hours after seeding, the cells were transfected with 50 ng each of pHMCA5-GFP and pHM5-U6-CMV-tetO2-CasRx-3xHA using Lipofectamine 3000. Forty-eight hours post-transfection, the cells were collected after dissociation to a single cell suspension in PBS containing 1% BSA. GFP-positive cells were isolated using a BD FACS Melody Cell Sorter (BD Biosciences, Franklin Lakes, NJ). A portion of the isolated cells were analyzed for assessing purity and the residual cells were immediately subjected to RNA preparation.

Statistical analysis

One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons post-hoc test and two-tailed Student’s t test was performed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA). Data are presented as means ± S.D.

Data and code availability

All data not included in the manuscript are available on request.

Acknowledgments

The authors thank Tomohito Tsukamoto (Graduate School of Pharmaceutical Sciences, The University of Osaka, Osaka, Japan) for advice. Images used in the graphical abstract were adapted from Servier Medical Art (https://smart.servier.com/), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). This study was supported by a Grant-in-Aid for Scientific Research (A) (23H00552) from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from the AMED (JP25ama121052). This work was also supported by JST SPRING (JPMJSP2138).

Author contributions

Y.O., investigation, methodology, writing – original draft, and writing – review & editing; S.K., investigation; E.S., supervision and writing – review & editing; A.A., investigation and methodology; F.S., supervision; K.S., supervision; H.M., conceptualization, supervision, funding acquisition, and writing – review & editing.

Declaration of interests

The authors declare no competing interests.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.omtn.2026.103057.

Supplemental information

Document S1. Figures S1–S16 and Tables S1–S5

mmc1.pdf^{(4.3MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(24MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S16 and Tables S1–S5

mmc1.pdf^{(4.3MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(24MB, pdf)}

Data Availability Statement

All data not included in the manuscript are available on request.

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PERMALINK

Adenovirus vector-mediated delivery of the CRISPR-CasRx system enables gene knockdown with suppressed collateral activity

Yuki Osakada

Sota Kidawara

Eiko Sakai

Akira Asayama

Fuminori Sakurai

Kahori Shimizu

Hiroyuki Mizuguchi

Abstract

Graphical abstract

Introduction

Results

Knockdown efficiency and collateral activity of CasRx, Cas13X.1, and PspCas13b delivered by plasmid transfection in human and mouse cell lines

Figure 1.

Generation of Cas13-expressing Ad vectors

Figure 2.

Knockdown efficiency and collateral activity of Ad-CasRx and Ad-PspCas13b in cells co-transduced with dual-reporter Ad vectors

Knockdown efficiency and collateral activity of Ad-CasRx and Ad-PspCas13b in the cells stably expressing the dual-reporter system

Figure 3.

Knockdown of Pcsk9 expression by Ad-CasRx

Figure 4.

In vivo knockdown of Pcsk9 by Ad CasRx

Figure 5.

Discussion

Materials and methods

Cells and reagents

Plasmids

Immunoblotting

Packaging cells for Ad vector production

Generation of SK-HEP-1 cells stably expressing Fluc and Gluc

Virus

Mouse experiments

Serum analysis

Real-time RT-PCR

In vitro luciferase assay

Measurement of cell viability

Fluorescence-activated cell sorting

Statistical analysis

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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