The expression of AKAP12 protein was decreased in livers of CCl₄-treated mice by 13% compared to oil controls (Figure 1A, left panel) without a change in Akap12 mRNA (Figure 1A, right panel). CCl₄ treatment induced the expression of HSC activation marker, Acta2 by 1.5-fold and its corresponding protein, α-SMA, by 6.4-fold compared to control (Figure 1A, Figure 1—source data 1). As evidenced by proximity ligation assay (PLA), the phosphorylation of AKAP12 was induced in desmin-positive HSCs of CCl₄ livers by fivefold compared to control (Figure 1B, Figure 1—source data 2). AKAP12 staining judged by ImageJ quantification (see Materials and methods) was decreased in CCl₄-treated liver by 16% compared to control (Figure 1B, Figure 1—source data 2) consistent with the western blot result (Figure 1A). The interaction of AKAP12 with HSP47 was inhibited by 54% despite a 3.9-fold increase in overall HSP47 levels in CCl₄ livers compared to control (Figure 1C, Figure 1—source data 3). A human liver fibrosis tissue array containing 16 liver fibrosis tissues and 11 normal tissues was stained with PLA probes for AKAP12 and HSP47 to detect their interaction. The interaction between AKAP12 and HSP47 was inhibited by 68% in human liver fibrosis tissue compared to normal (Figure 1D, Figure 1—figure supplement 1). This was associated with a 20% decrease in total AKAP12 staining and a 3.8-fold increase in HSP47 staining in liver fibrosis compared to normal (Figure 1D). Post hoc analysis of Figure 1A–D is provided in Figure 1—source data 4.

The phospho-peptide map of AKAP12 protein from Day 7 culture-activated human or mouse HSCs was compared to that of Day 0 quiescent HSCs or normal hepatocytes (Table 1). A peptide region containing 5S/T phospho-sites exhibited increased phosphorylation in Day 7 activated HSCs but not in Day 0 HSCs or hepatocytes (Table 1—source data 1). These activation-responsive phospho-sites were conserved in mouse and human (Table 1). The interaction between AKAP12 and HSP47 was reduced by 40% after 3 days and by 86% after 6 days of HSC culture-activation compared to Day 0 (Figure 2A, Figure 2—source data 1). This was associated with a corresponding induction in the levels of α-SMA up to 5.8-fold by Day 6 compared to Day 0 (Figure 2A, Figure 2—source data 1). Day 5 activated human HSCs were transfected with CRISPR small guide RNA (sgRNA) and donor RNA (see Key resource table) to delete the five AKAP12 phosphorylation sites by homology-directed repair (HDR) as described under Materials and methods. Genomic DNA PCR from CRISPR edited (HDR) cells using deletion-specific primers (see Key resource table) resulted in a 261-bp amplicon that was not amplified in wild-type (WT) cells or cells treated with SaCas9 (Staphylococcus aureus CRISPR-associated protein) alone (Figure 2B, original gel shows four experiments). The interaction between AKAP12 and HSP47 in CRISPR-edited HSCs (HDR) was induced by 2.5-fold compared to WT cells (Figure 2C, original blot developed with anti-mouse IgG is shown in Figure 2—source data 2). This was associated with a 40% decrease in α-SMA levels, demonstrating that AKAP12 phospho-site editing inhibited HSC activation (Figure 2C). The overall level of HSP47 decreased by 25% whereas the level of AKAP12 protein remained unchanged after HDR (Figure 2C). Deletion of phospho-sites in mouse HSCs resulted in a 422-bp deletion-specific amplicon (Figure 2—figure supplement 1, Figure 2—figure supplement 1—source data 1). Like human HSCs, mouse HSCs also exhibited increased AKAP12-HSP47 interaction after AKAP12 phospho-site editing (Figure 2—figure supplement 1, Figure 2—figure supplement 1—source data 2). Reversal of HSC activation by AKAP12 editing was determined by examining vitamin A auto fluorescence (Senoo et al., 2010). Cultured human HSCs at Day 0 exhibited strong vitamin A autofluorescence that was reduced in Day 5 activated HSCs (Figure 2D, three independent experiments are shown). AKAP12 editing in Day 5 HSCs restored the loss of vitamin A fluorescence compared to Day 5 HSCs or Day 5 HSCs+SaCas9 alone (Figure 2D). HSP47 is an ER-resident chaperone (Kawasaki et al., 2015). A weak PLA signal of AKAP12-HSP47 interaction co-localized with the ER marker, calreticulin in activated (WT) HSCs (Figure 2E, left panel). However, upon CRISPR-editing (HDR), a strong AKAP12-HSP47 PLA signal co-localized with calreticulin in the ER (Figure 2E, left panel, Figure 2—source data 3). We examined whether HSP47’s collagen-chaperoning activity was regulated by AKAP12 phospho-site editing. Our results show that the collagen-HSP47 PLA signal strongly co-localized in the ER of activated HSCs (WT) (Figure 2E, right panel). CRISPR-editing of AKAP12 (HDR) reduced the collagen-HSP47 interaction significantly by 65% compared to WT cells (Figure 2E, right panel, Figure 2—source data 3). Post hoc analysis of Figure 2A, C and E is provided in Figure 2—source data 4.

Kinase-prediction software was used to predict that out of the five AKAP12 activation-responsive phospho-sites, two serine residues (S687/S688) were strongly predicted substrates of PKCα kinase with a consensus of [S/T]-X-R/K whereas one threonine (T675) could not be assigned a kinase (Supplementary file 1). S676/S678 sites were also PKCα sites but shared consensus sites with calmodulin kinase (CAMK). The overall confidence of prediction for the S676/S678 sites was less than that of S687/S688 sites. In vitro kinase assay followed by phostag gel analysis revealed that phosphorylation of AKAP12 was significantly enhanced in the presence of active PKCα enzyme compared to kinase negative controls (Figure 3A). Mutations of AKAP12 S676/S678 to alanine modestly reduced the phosphorylation of biotinylated recombinant AKAP12 whereas S687A/S688A mutation dramatically suppressed the phostag shift of AKAP12 (Figure 3A). The mutation seemed to completely inhibit the phospho-band. Since other phosphorylation events could also cause the shift, we repeated the experiment to see whether this complete suppression was reproducible. In an additional experiment (Figure 3—source data 1), we observed that the S687/S688A mutation suppressed but did not always wipe out the phospho-shift. Also, in some experiments, we observed the -kinase control had a faint phospho-signal. The recombinant protein produced by RRLs in an in vitro translated system may have baseline phosphorylation as reported in the manufacturer’s protocol (TNT Coupled Transcription/Translation system, Promega). Direct binding was observed between biotinylated AKAP12 and HSP47 in a recombinant system in the absence of active PKCα (Figure 3B, Figure 3—source data 2). Presence of PKCα inhibited the interaction between AKAP12 and HSP47 (Figure 3B). To evaluate whether phosphorylation of AKAP12 by PKCα in HSCs would regulate AKAP12’s scaffolding activity, cells were treated with Prkca siRNAs (A or B). Silencing Prkca by 74% with siRNA-A increased AKAP12-HSP47 interaction by threefold whereas a 90% knockdown caused by siRNA-B enhanced AKAP12-HSP47 interaction by eightfold compared to negative control siRNA (Figure 3C, Figure 3—source data 3). HSP47 levels remain unchanged by siRNA treatments (Figure 3C). Post hoc analysis of Figure 3C is provided in Figure 3—source data 4.

The Akap12 exon 3 contains sequences corresponding to the activation-responsive phospho-sites of AKAP12 protein. To perform gene editing of this region specifically in HSCs of mouse liver, two different CRISPR HDR approaches were used (Figure 4A). A PDEL donor was used to delete the AKAP12 phospho-sites whereas each S or T phospho-site was mutated to A using a PMUT donor. Two unique sgRNAs specific for the region around the phospho-sites along with the donor (Figure 4A, Key resource table) were cloned into AAV vectors (Figure 4B, left panel). To perform CRISPR editing in HSCs of mouse liver, the SaCas9 enzyme was cloned into AAV vector under control of two different HSC-specific promoters (Glial fibrillary acidic protein, GFAP or Lecithin retinol acyltransferase, LRAT) (Puche et al., 2013; Lee et al., 2020). AAV vectors were injected into mice during oil or CCl₄ administration according to the plan in Figure 4B, right panel. To evaluate the HSC specificity of GFAP-SaCas9 mediated CRISPR (CR) editing compared to that of an empty vector (EV) control (see Materials and methods), genomic DNA of HSCs or hepatocytes isolated from livers of oil+EV, oil+CR, CCl₄+EV, and CCl₄+CR groups was subjected to multiplex PCR with PDEL forward and reverse primers and a PDEL deletion-specific primer (see Key resource table). Oil+EV or CCl₄+EV HSCs or hepatocytes gave a 298-bp amplicon in this multiplex PCR. Oil+CR or CCl₄+CR groups resulted in 298 bp WT and 256 and 154 bp mutated amplicons due to complementarity with the deletion-specific primer (Figure 4C, Figure 4—source data 1). PCR with deletion-specific primers did not amplify the 256 or 154 bp mutant region in hepatocytes, indicating that CRISPR-editing using an HSC promoter-specific SaCas9 occurred in HSCs but not hepatocytes (Figure 4E, Figure 4—source data 2). A specific primer to detect PMUT could not be designed, hence PMUT specificity was evaluated by next-generation amplicon sequencing (NGS) as in Figure 4G. The efficiency of AAV transduction by PDEL or PMUT donor was evaluated under oil or CCl₄ conditions by immunostaining of SaCas9 enzyme with HSC marker, desmin or hepatocyte marker, and albumin. The GFAP-driven SaCas9 enzyme strongly co-localized with desmin-positive HSCs in PDEL or PMUT transduced livers (Figure 4D) but not with albumin-positive hepatocytes in the liver (Figure 4F). The efficiency of transduction of PDEL or PMUT as calculated by the SaCas9 count per desmin area was not significantly different between PDEL and PMUT under either oil or CCl₄ conditions (Figure 4D). CCl₄ exposure increased the overall numbers of desmin-positive HSCs (Figure 4D) due to increased activation and proliferation (Fujii et al., 2010). Post hoc analysis of Figure 4D and F is provided in Figure 4—source data 3. The efficiency of gene editing was tested by using a 298-bp amplicon from HSCs or hepatocytes of GFAP-SaCas9 CRISPR livers (Figure 4G). On-target and off-target base changes were analyzed by comparing the target read sequences to the reference sequence of WT Akap12 amplicon as described under Materials and methods. For the PDEL CRISPR, oil+CR HSCs exhibited 30% mutant reads compared to the total reads whereas CCl₄+CR HSCs exhibited 60% mutant reads compared to total (Figure 4G, top panel, Supplementary file 2). Oil+EV or CCl₄+EV HSCs did not contain any mutant reads (Figure 4G, top panel). For the PMUT CRISPR, oil+CR HSC exhibited 3% mutant reads and CCl₄+CR exhibited 12.5% mutant reads compared to total reads (Figure 4G, bottom panel, Supplementary file 3). Hepatocytes from the CR groups did not exhibit any PDEL or PMUT sequence reads (Figure 4G, Supplementary files 2 and 3). The percentage of base changes outside the target region between the two sgRNA sites (Figure 4A) was less than 5% in most cases (Figure 4G). Like GFAP-SaCas9, PDEL CRISPR was also observed with LRAT-SaCas9 in HSCs (Figure 4—figure supplement 1) but not hepatocytes (Figure 4—figure supplement 1). The LRAT-driven SaCas9 expression in desmin-positive HSCs was lower in the CCl₄ group compared to oil (Figure 4—figure supplement 1) and not significant in hepatocytes (Figure 4—figure supplement 1). The CRISPR deletion efficiency using LRAT-SaCas9 in HSCs of oil+CR group was 45% whereas that of the CCL₄+CR group was 30% of total reads (Figure 4—figure supplement 1, Supplementary file 2).

At gross level, CCl₄ administration for five weeks reduced the body weight of mice by 20% (Figure 5A) and increased the liver to body weight ratio by 1.25-fold compared to oil (Figure 5B). AKAP12 phospho-editing by GFAP-SaCas9 in normal mice (oil+CR) did not alter body or liver weight compared to oil+EV (Figure 5A). However, AKAP12 phospho-editing in CCL₄ mice (CCl₄+CR) normalized CCL₄+EV-mediated alterations in body weight and liver/body weight to that of oil+EV levels (Figure 5A, Figure 5B). Histologically, control mice (oil) had a normal hepatic cord pattern around the central vein, whereas fatty vacuolar changes and disorganized hepatic lobular structure with centrilobular fibrosis were observed in CCl₄ livers (Figure 5C, PDEL-top panel, PMUT-bottom panel) as referenced previously (Wang et al., 2012). AKAP12 phospho-editing by PDEL in CCl₄ mice (CCl4+CR PDEL) dramatically reduced the CCl₄-induced histological distortions compared to CCl₄+EV (Figure 5C, top panel). AKAP12 PMUT editing also suppressed the histological changes but less dramatically compared to PDEL (Figure 5C, bottom panel), hematoxylin and eosin (H&E) staining for individual PDEL and PMUT experiments is shown in Figure 5—source data 1. Picosirus red staining of CCl₄ livers showed increased collagen deposition that was substantially reduced when mice were administered AKAP12 phospho-editing vectors (Figure 5D). Sirius red staining for individual PDEL and PMUT experiments is shown in Figure 5—source data 2. The hydroxyproline content of collagen was increased 2.4-fold in CCl₄ livers compared to oil+EV and normalized by AKAP12 PDEL (Figure 5E, top panel). PMUT phospho-editing inhibited CCl₄-mediated induction but did not completely normalize hydroxyproline content compared to oil+EV or oil+CR (Figure 5E, bottom panel). CCl₄ administration caused an 8- to 13-fold induction in liver injury as measured by ALT/AST levels (Figure 5F). AKAP12 phospho-editing by PDEL or PMUT in control mice (oil+CR) did not affect the levels of ALT/AST (Figure 5F). AKAP12 phospho-editing by PDEL or PMUT in CCl₄ mice (CCl4+CR PDEL or PMUT, left and right panels, respectively) dramatically reduced the ALT/AST level by 75–80% compared to CCl₄+EV (Figure 5F). Compared to CCl₄+CR PDEL, CCl₄+CR PMUT ALT levels were not normalized to oil+CR levels but were statistically higher than that of oil+CR group (Figure 5F, right panel). Post hoc analysis of Figure 5A, B, E and F is presented as Figure 5—source data 3. LRAT-SaCas9 directed PDEL-CRISPR also resulted in higher body weight, lower liver/body weight ratio, and suppression of CCl₄-induced histological changes like that of GFAP-SaCas9.

The AKAP12-HSP47 scaffold was reduced in livers of CCl₄+EV mice compared to oil controls (Figure 6A and B). PDEL-CRISPR or PMUT-CRISPR editing in CCl₄ mice restored the drop in the AKAP12-HSP47 interaction caused by CCl₄ (Figure 6A, PDEL; Figure 6B, PMUT, Figure 6—source data 1, PDEL; Figure 6—source data 2, PMUT). AKAP12 phospho-editing dramatically inhibited CCl₄-mediated HSC activation as evidenced by a drop in α-SMA levels (Figure 6A and B). In conjunction with restoration of AKAP12-HSP47 scaffold, the increased interaction between collagen and HSP47 upon CCl₄ exposure was inhibited by AKAP12 phospho-editing (Figure 6C). Co-immunoprecipitation of collagen with HSP47 antibody yielded non-specific bands at positions above the collagen position in all samples including IgG control. The original uncropped blot is shown in Figure 6—source data 3. AKAP12 PDEL or PMUT phospho-editing also inhibited the increase in Col1a1 and Acta2 mRNA levels caused by CCl₄ exposure (Figure 6D). Col1a1 levels were normalized to oil+EV levels by both PDEL and PMUT CRISPRs (Figure 6D, left and right panels). However, compared to PDEL, PMUT CRISPR reduced but did not completely normalize Acta2 levels to control (oil+EV) state (Figure 5D, left and right panel). PLA staining showed that the AKAP12-HSP47 scaffold was localized with desmin-positive HSCs under normal (oil) conditions (Figure 6D). A drop in AKAP12-HSP47-desmin co-localization was observed upon CCl₄ exposure that was restored by AKAP12 PDEL or PMUT phospho-editing (Figure 6D). Post hoc analysis for Figure 6A–E is presented as Figure 6—source data 4.

To determine how HSC-specific AKAP12 phospho-editing reduced overall liver injury and modulated collagen mRNA levels upon CCl₄ exposure, we performed proteomics analysis of HSCs and livers isolated from oil+EV, oil+CR, CCl₄+EV, or CCl₄+CR groups to compare the molecular changes under these conditions. Proteomics analysis revealed alterations in several proteins in CCl₄ HSCs as well as total liver that were regulated by AKAP12 HSC-specific phospho-editing (Supplementary file 4). Ingenuity pathway analysis (IPA) of these proteins identified two top scoring pathways, the ER stress response and UPR, that were significantly dysregulated by CCl₄ and were normalized upon AKAP12 phospho-editing (Supplementary file 4). The proteomics analysis of HSCs showed an induction in BIP/GRP78, an ER stress sensor (Sepulveda et al., 2018), in CCL₄-treated group (Figure 7A, Supplementary file 4). We confirmed the proteomics by western blotting. GAPDH-normalized densitometries from individual experiments are presented in Figure 7—figure supplement 1. HSCs isolated from CCl₄ livers showed increased BIP expression (Figure 7B). However, even though the proteomics analysis showed inhibition of CCl₄-induced BIP levels by AKAP12 phospho-editing (Figure 7A), we could not confirm this effect by western blotting (Figure 7B). Since BIP is a known collagen chaperone (Sepulveda et al., 2018), we examined its interaction with collagen in our CRISPR model. BIP exhibited increased interaction with collagen in the CCl₄+EV HSCs compared to oil+EV HSCs and AKAP12 phospho-editing strongly inhibited the BIP-collagen interaction in HSCs (Figure 7B). IRE1α, a UPR component that binds to HSP47 and becomes phosphorylated during ER stress (Sepulveda et al., 2018), exhibited increased interaction with HSP47 in CCL₄ HSCs that was inhibited by AKAP12 phospho-editing (Figure 7B). The IRE1α-HSP47 interaction was further confirmed in desmin-positive HSCs of the CRISPR model by PLA staining (Figure 7—figure supplement 2). CCl₄-mediated IRE1α phospho-activation (S724 phosphorylation) was strongly inhibited by AKAP12 phospho-editing without a change in total IRE1α levels (Figure 7B). Furthermore, two pathways, P38MAPK and SMAD2/3 that are known to be induced in HSCs through IRE1α activation (de Galarreta et al., 2016) were also suppressed by AKAP12 phospho-editing (Figure 7B). The proteome of CCl₄-exposed livers exhibited increased ER stress and UPR signaling components that were modulated by AKAP12 HSC-specific phospho-editing (Figure 7A, Supplementary file 4). BIP levels by western blotting were induced in CCl₄ livers and inhibited by AKAP12 phospho-editing, confirming the proteomics result (Figure 7C). Like the proteomics data, we did not find any change in total IRE1α expression. However, phospho-activated IRE1α was suppressed by AKAP12 phospho-editing in total liver (Figure 7C). Since ER stress induces inflammatory signals in different systems (Maiers and Malhi, 2019), we examined whether the HSCs from our CRISPR mouse model exhibited altered inflammatory signaling upon AKAP12 phospho-modulation. Out of the known HSC cytokines, we found the pro-inflammatory cytokine, IL-17, IL-6, and IL-β to be strongly induced in CCl₄-HSCs whereas AKAP12-phospho-edited HSCs suppressed their expression (Figure 7D). On the other hand, an anti-inflammatory cytokine, IL-10 was suppressed in HSCs by CCl₄ administration, and its expression was restored by AKAP12 phospho-editing (Figure 7D). To examine whether ER stress modulation within activated HSCs was transmitted to other liver cell types, we evaluated crosstalk between HSCs and hepatocytes in a co-culture system where AKAP12 was CRISPR-edited. Co-culture with activated HSCs induced the ER stress response markers BIP and induced IRE1α phosphorylation in hepatocytes compared to co-culture with quiescent HSCs (Figure 7E). Co-culture with activated HSCs in which AKAP12 was phospho-edited (CR) reduced the ER stress signal in hepatocytes compared to activated HSCs alone whereas hepatocytes co-cultured with quiescent HSCs with CR did not exhibit any alteration in ER stress markers compared to WT (Figure 7E). Original blots for Figure 7B–E are shown in Figure 7—source data 1 and Figure 7—source data 4. Post hoc analysis for Figure 7B–E is presented in Figure 7—source data 5.

Primary human HSCs purchased from ScienCell Incorporation (CA) were cultured on plastic dishes for 6 hr (Day 0) or further cultured till activation (Days 5–7). Mouse HSCs or hepatocytes were isolated from 3 to 4 months old C57BL/6 mice according to our previously established protocols (Ramani et al., 2018). Mouse HSCs were culture-activated on plastic dishes like human HSCs.

AKAP12 was immunoprecipitated from HSCs or hepatocytes using an AKAP12 antibody-conjugated protein A/G column (Thermo Fisher Scientific). The AKAP12 beads were submitted to Applied Biomics, CA for phospho-peptide mapping. Tryptic peptides were enriched for phospho-peptides and processed for detection of a phospho-site by mass spectrometry. Phosphorylated residues were confirmed by mass spectrometry peak showing the neutral loss of phosphate that was detected from peak shifts on MS/MS spectrum (Table 1—source data 1). The observed mass of a phospho-peptide was reduced by 98 Da if a single serine/threonine showed a neutral loss of phosphate.

CRISPR-Cas9 mediated gene editing at the AKAP12 gene locus (exon 3) to delete the region of its activation-responsive phospho-sites was performed by HDR. A 22-bp small guide RNA sequence (sgRNA) upstream of a protospacer adjacent motif (PAM- 5′-GTGGAT-3′) recognized by saCas9 (PAM consensus-NNGRRT where N=any nucleotide, R=A or G) (Xie et al., 2018), was designed and synthesized using the Edit-R CRISPR system (Horizon Discovery, CO) (human guide sequence, Key resource table). The CRISPR design tool was used to determine the sgRNA whose sequence is unique compared to the rest of the genome to avoid off-target effects. A donor RNA to delete the phospho-region was designed and synthesized using the Edit-R HDR donor designer system (Horizon) (human PDEL HDR donor, Key resource table). The sgRNA was stabilized by 2′-O-methyl nucleotides and phosphorothioate linkages in the backbone on both the 5′ and 3′ ends and the HDR donor was stabilized by phosphorothioate linkages on both ends to improve functionality during transfection. Cultured cells were co-transfected with a commercially available plasmid, AAV6-GFAP-saCas9, containing the SaCas9 gene under control of the GFAP promoter (Vector Biolabs, PA), sgRNA and HDR donor RNA using the DharmaFECT Duo Transfection Reagent that allows co-transfection of RNA and DNA (Horizon). Cells with transfection reagent alone or SaCas9 plasmid alone +transfection reagent were used as controls. CRISPR designs for mouse HSCs were performed as above for human with mouse guide sequence #1 and mouse PDEL HDR donor (Key resource table). After 48–72 hr of transfection, genomic DNA from human or mouse HSCs was amplified by multiplex PCR using two primers to amplify the region around the deletion site and a third deletion-specific primer to detect HDR-mediated gene editing.

Activated human HSCs (0.3 million cells per well of six-well plate) were reverse transfected with a universal negative control (Cat #4404021), Prkca (Cat #s11092), or Prkca B (Cat #s11094) silencerselect siRNA (Thermo Fisher Scientific, IL) using the lipofectamine RNAiMAX reagent as we described previously (Ramani et al., 2018).

About 12-week-old C57BL/6 male mice were injected intraperitoneally with CCl₄ (HPLC grade, Cat #270652, Sigma-Aldrich, diluted 1:3 in mineral oil) or mineral oil (control) at 1 µl/gram body weight bi-weekly for 5 weeks. All procedures for the care and use of mice were approved by the Institutional Animal Care and Use Committee at Cedars-Sinai Medical Center (CSMC).

HDR-based gene editing in control or CCl₄ mice was performed according to the scheme in Figure 4A and B. Two 22-bp sgRNA sequences upstream of a saCas9 PAM (Xie et al., 2018), were designed using the Edit-R CRISPR system (Horizon Discovery). Off-target analysis for the two sgRNA was performed using the algorithm from the Benchling (Biology Software-(2022) retrieved from https://benchling.com) (Supplementary file 5). The two sgRNA sequences were cloned into a single AAV6 vector under the control of a U6 promoter by the cloning service available from Vector builder Inc, IL. An AAV6 vector containing a non-targeting sgRNA was used as an EV control. The sequence corresponding to a PDEL or PMUT donor with 500 bp flanking either side of the target region was cloned into a separate AAV6 vector. The PAM sequence in these donors was mutated to prevent re-cleavage by SaCas9 after HDR. The AAV6-GFAP-SaCas9 vector (Vector Biolabs) was used for HSC-specific gene editing. In addition, another AAV6-LRAT-SaCas9 vector was prepared by cloning the mouse LRAT promoter (Accession ID: NM_023624) upstream of SaCas9 (Vector Builder). AAV6 particles of the sgRNA construct, EV construct, PDEL/PMUT donors, and GFAP/LRAT-SaCas9 were purified using Vector builder’s AAV production service. For each viral vector, titer was determined by real-time PCR using primers specific for the AAV inverted terminal repeats (ITRs). A titer of 1–2×10¹³ genome copies (GCs)/ml was achieved for each AAV. All vectors tested negative for mycoplasma contamination. EV or sgRNA vectors along with PDEL or PMUT donors and GFAP or LRAT SaCas9, were injected into tail vein of mice at 10¹¹ GC/vector in a volume of 100 µl phosphate-buffered saline. Viral vectors were injected into oil or CCl₄ mice during the second and fourth week of oil or CCl₄ administration (Figure 4B). The HSC specificity of CRISPR was determined by SaCas9 immunofluorescence as described under the Immunostaining section. The efficiency of CRISPR editing in HSCs and hepatocytes of gene-edited livers was evaluated by NGS. A 298-bp PCR product was amplified from genomic DNA using primers that recognized regions upstream and downstream of the site of AKAP12 deletion or mutation. Amplicons were purified from gels and submitted to Azenta Life Sciences Inc, CA. for performing NGS. Briefly, Illumina adaptor sequences (FW: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′, REV: 5′-GACTGGAGTTCAGACGTGTGCTCTTCCGA TCT-3′) were added to the amplicons and sequenced by Azenta Illumina platform sequencers. The WT and mutant or deletion mutant reads were counted from each sample and the efficiency of editing was the percentage of edited reads (PDEL or PMUT) versus the total reads. Frequencies of on-target and off-target base changes were analyzed by comparing the target reads to reference reads corresponding to the WT Akap12 amplicon between the two sgRNA sequences (Figure 4A). Within this region, any mismatches other than PDEL or PMUT were considered as off-targets. The mismatches to the reference were observed mainly outside the target region at a frequency of 5% or less (Figure 4G).

The human tissue array (Cat #XLiv086-01) in the form of paraffin-embedded tissues was purchased from the human tissue biorepository, US Biolabs Inc, MD. Arrays were stained by immunostaining as described below.

Total RNA from cells or tissues was reverse transcribed to cDNA using M-MLV reverse transcriptase (Nxgen). CDNA was subjected to quantitative RT-PCR using TaqMan probes for mouse Akap12, Col1a1, Acta2, and the housekeeping gene, Gapdh (mouse) (Life Technologies) (Ramani et al., 2018). The PCR profile was: initial denaturation: 95°C for 3 min, 45 cycles: 95°C, 3 s; 60°C, 30 s. The cycle threshold (Ct value) of the target genes was normalized to that of control gene to obtain the delta Ct (ΔCt). The ΔCt was used to find the relative expression of target genes according to the formula: relative expression=2^−ΔΔCt, where ΔΔCt=ΔCt of target genes in experimental condition − ΔCt of target gene under control condition.

Total protein extract was processed for immunoprecipitation by incubating 200 µg of pre-cleared protein with 2 µg of antibody as we described previously (Ramani et al., 2018). Immunoprecipitated protein was processed for western blotting as previously published (Ramani et al., 2018) and developed with Clean-blot IP detection reagent (HRP) (Thermo Fisher Scientific, IL). Antibodies used for western blotting are listed in Key resource table.

UV-excited autofluorescence of human HSCs was captured by fluorescence microscopy using a Keyence BZ-X710 inverted fluorescent microscope (Itasca, IL) as we described previously (Ramani et al., 2018).

An expression vector (pReceiver-WG16) containing the human AKAP12 gene under control of the T7 promoter was purchased from Genecopoiea, MD and mutated at AKAP12’s activation-responsive sites (S/T to A mutations) using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, CA) as we described previously (Ramani et al., 2015). Mutations were detected by sequencing the clones at the Azenta DNA sequencing facility using an AKAP12 gene-specific primer (5′-GAGAAGGTGTCACTCCC-3′).

The T7-AKAP12 vector or its mutants were in vitro translated using the non-radioactive TNT Coupled Transcription/Translation system containing rabbit reticulocyte lysate (RRL) and a biotin-lysyl tRNA according to the manufacturer’s instructions (Promega, WI) to incorporate biotin label into the translated AKAP12 protein. Biotinylated AKAP12 was purified from the RRL components using a biotin-antibody column. Biotinylated AKAP12 or its mutants (5 µl) were used as a substrate for PKCα in a 25-µl in vitro kinase reaction using 100 ng of active recombinant PKCα enzyme (MilliporeSigma, MA), 5 µl of a lipid activator (MilliporeSigma; 20 mM MOPS, pH 7.2, 25 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 1 mM CaCl2), 3 µl of Mg^2+/ATP cocktail (MilliporeSigma, 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM Na₃VO₄, 1 mM dithiothreitol, 75 mM MgCl₂, and 0.5 mM ATP) and 2.5 µl of 20 mM Hepes-NaOH buffer, pH 7.6. The reaction was carried out at 30°C for 2 hr. The kinase reaction was run on a zinc phostag gel containing 15 µM phostag gel (Fujifilm Wako Chemicals, VA) to separate phosphorylated form of AKAP12 from its unphosphorylated counterparts as we described earlier (Ramani et al., 2015). Membranes were probed with streptavidin-HRP (Key resource table) to detect biotinylated AKAP12. Biotin antibody was conjugated to protein A/G plus agarose columns using a coupling buffer according to the crosslink immunoprecipitation kit (Thermo Fisher Scientific) followed by binding of recombinant biotinylated AKAP12. The columns were treated with recombinant HSP47 protein in the absence or presence of active PKCα enzyme. Bound proteins were eluted from the washed column using elution buffer from the crosslinking immunoprecipitation kit (Thermo Fisher Scientific) and run on gels along with biotinylated AKAP12 as input and antibody-bound protein A/G beads as IgG controls. Blots were incubated with HSP47 antibody followed by Clean-blot IP detection. Reverse IP was done by following the same protocol using HSP47 antibody columns treated with biotinylated AKAP12 followed by detection with streptavidin-HRP. Recombinant HSP47 input was purchased from Prospec protein specialists, NJ.

For immunocytochemical procedures, cells were fixed with paraformaldehyde and then permeabilized with Triton-X 100 before antibody staining. For immunohistochemical analysis, tissues were de-paraffinized and antigen retrieval was performed using the citrate-based antigen unmasking solution (Vector Laboratories, CA). For phospho-detection using PLA, primary AKAP12 or phospho-serine (PSer) antibodies (see Key resource table) were directly conjugated to PLA minus or plus complementary oligonucleotide arms (PLA minus, Catalog no. DUO92010; PLA plus, Catalog no. DUO92009, MilliporeSigma) according to our previously published protocol (Ramani et al., 2018). To examine protein-protein interactions in cells or tissues, samples were incubated with the antibodies for the interacting targets at 4°C overnight (AKAP12-HSP47, HSP47-collagen). After washing the unbound antibodies, samples were further incubated overnight with secondary antibodies (rabbit or mouse) that were bound to PLA plus or minus complementary probes (MilliporeSigma, Key resource table). The PLA probes were ligated when the proteins were in proximity due to their interaction giving a fluorescent signal as we previously reported (Ramani et al., 2018). To evaluate the localization of interacting partners, co-immunostaining of the PLA signals was done with HSC (desmin) or subcellular compartment (calreticulin ER) marker antibodies. Marker antibodies were detected by Alexa fluor green rabbit or mouse secondary antibodies (Abcam, Key resource table). Co-localization of SaCas9 with desmin or albumin markers in liver tissue was detected by Alexa fluor secondary antibodies (see Key resource table). AKAP12 expression in tissues was detected using the mouse HRP/DAB detection immunohistochemistry kit (Cat #ab64264, Abcam).

Liver sections fixed with 10% neutral formalin were processed for paraffin embedding, sectioning, H&E, and picrosirius red staining (collagen) using the services provided by the liver histology core of the University of Southern California research center for liver diseases (NIH grant P30 DK048522).

The hydroxyproline content of tissue was measured following the protocol from the hydroxyproline assay kit (Cell Biolabs Inc, CA). Briefly, 10 mg of liver tissue was homogenized, and acid hydrolysis was done with 12 N HCl. Hydrolyzed samples were treated with chloramine T to convert the hydroxyproline to a pyrrole. Ehrlich’s reagent or 4-(Dimethylamino) benzaldehyde added to the pyrrole reacted with it to produce a chromophore whose absorbance could be read at 540–560 nm. The content of hydroxyproline in the tissue sample was determined by comparison to a hydroxyproline standard from the kit that was processed like the unknown sample.

ALT and AST levels from plasma of mice were measured with the ALT and AST colorimetric activity assay kits (Cayman Chemical, MI). ALT activity was measured by monitoring the rate of NADH oxidation in a coupled reaction using lactate dehydrogenase (LDH). The NADH to NAD+ oxidation caused a decrease in A340 nm absorbance. The rate of decrease (ΔA340/min) is directly proportional to the ALT activity. AST activity was measured by the rate of NADH oxidation in the presence of malate dehydrogenase. NADH to NAD+ conversion caused a decrease in A340 nm absorbance. LDH was added to the AST reaction to prevent interference from endogenous pyruvate in the plasma. The ΔA340/min for both ALT and AST were converted to units/L by dividing the ΔA340 values by the NADH extinction coefficient and multiplying by the sample dilution factor as per the protocol instructions (Cayman).

Total protein from liver or HSCs was subjected to mass spectrometry-based proteomics analysis by the services of Poochon proteomics solutions, MD. The Nanospray LC/MS/MS analysis of tryptic peptides for each sample was performed sequentially with a blank run between each two sample runs using a Thermo Scientific Orbitrap Exploris 240 Mass Spectrometer and a Thermo Dionex UltiMate 3000 RSLCnano System. Peptides from trypsin digestion were loaded onto a peptide trap cartridge at a flow rate of 5 μl/min. The trapped peptides were eluted onto a reversed-phase Easy-Spray Column PepMap RSLC, C18, 2 μM, 100 A, 75 μm×250 mm (Thermo Fisher Scientific, CA) using a linear gradient of acetonitrile (3–36%) in 0.1% formic acid. The elution duration was 110 min at a flow rate of 0.3 μl/min. Eluted peptides from the Easy-Spray column were ionized and sprayed into the mass spectrometer, using a Nano Easy-Spray Ion Source (Thermo Fisher Scientific) under the following settings: spray voltage, 1.6 kV, Capillary temperature, 275°C. Other settings were empirically determined. Raw data files were searched against mouse protein sequences database using the Proteome Discoverer 1.4 software (Thermo Fisher Scientific) based on the SEQUEST algorithm. Carbamidomethylation (+57.021 Da) of cysteines was set as fixed modification, and Oxidation/+15.995 Da (M), and Deamidated/+0.984 Da (N, Q) were set as dynamic modifications. The minimum peptide length was specified to be five amino acids. The precursor mass tolerance was set to 15 ppm, whereas fragment mass tolerance was set to 0.05 Da. The maximum false peptide discovery rate was specified as 0.05. The resulting Proteome Discoverer Report contains all assembled proteins with peptides sequences and peptide spectrum match counts (PSM#). The PSM count is a measure of the abundance of the protein.

Western blotting data were quantified by densitometry of blots using the ImageJ software (NIH). PLA staining and immunofluorescence data were analyzed in a blinded manner by two individuals and quantified using ImageJ according to published protocols (López-Cano et al., 2019). Scatter bars showing individual experimental points and their means were plotted using GraphPad Prism 9.3.0, GraphPad software. Biologically independent replicates combined from at least three individual experiments were represented as mean ± standard error (mean ± SE). Statistical analysis was performed using two-tailed Student’s t-test for paired comparisons and one-way ANOVA (GraphPad Prism) for comparing differences between multiple groups. Significance was defined as p<0.05. Tukey HSD post hoc test for each comparison is shown as source data.