Hereditary tyrosinemia type I mice
All animal study procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Massachusetts Medical School. HT-I mice were kindly provided by Dr. M. Grompe and maintained on C57 background for the FahPM/PM strain, and on a 129 background for Fahneo/neo. Mice were kept on 10 mg/L 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC; Sigma-Aldrich, Cat. No. PHR1731-1G) in drinking water when indicated. Male and female mice (8 to 15 weeks old) were used, and their body weights were recorded every 1–3 days. Moribund mice with more than 10% body weight loss were humanely euthanized, and sera and liver tissues were harvested for genome editing analyses. Tissues were pulverized in liquid nitrogen and aliquots of tissue powder (~30 mg) were used to extract total DNA [using the DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturer’s protocol], as well as total RNA and total protein.
Mucopolysaccharidosis type I mice
Homozygous MPS-I IduaW392X mice93 were purchased from the Jackson Laboratory (Stock No. 017681) and used to breed neonatal IduaW392X pups for rAAV injections. Postnatal day 1 mice were injected with 4 × 1011 gc of rAAV:HDR:uncleaved, rAAV:HDR:cleaved, or control vectors via the facial vein. Five weeks after injection, blood was collected via cardiac punch, and mice were transcardially perfused with ice-cold PBS. Tissues were immediately dissected, snap-frozen in liquid nitrogen, and stored at −80 °C. Both female and male animals were used. All animal procedures were reviewed and approved by the IACUC at the University of Massachusetts Medical School and performed in compliance with all relevant ethical regulations.
Cloning of rAAV plasmids
All rAAV vectors were based on our previously published rAAV plasmid [Nme2Cas9_AAV; Addgene plasmid #119924] design24, with modifications to increase the cargo capacity (Supplementary Fig. 1). Human-codon-optimized Neisseria meningitidis DE10444 Cas9 (Nme2Cas9) is under the expression of a U1a promoter, with one SV40 nuclear localization signal (NLS) on the N terminus and one nucleoplasmin NLS on the C terminus, followed by a short poly(A) signal59. Shortened sgRNA Nme.sgRNA-121 was synthesized as a gblock for cloning. Target spacer sequences were inserted by digesting the plasmid with the BspQI restriction enzyme, and the annealed spacer sequence carrying appropriate BspQI overhang sequences were ligated. This plasmid is available on Addgene (pEJS1089: mini-AAV:sgRNA.Nme2Cas9; Addgene # 159536).
Cloning of dual-guide rAAV:Nme2Cas9 plasmids
A second U6 promoter (from human U6 for designs 1, 2, and 4; and from mouse U6 for design 2) with a Nme.sgRNA-121 expression cassette was generated as a gblock (IDT) and cloned into the minimized rAAV:Nme2Cas9 plasmid to create dual-sgRNA designs 1–4 (Fig. 1a). The two configurations that enabled efficient packaging of full-length vector genomes (designs 1 and 4) are available on Addgene (pEJS1099: Dual-sgRNA:Design 4, Addgene #159537; pEJS1096: Dual-sgRNA:Design 1, Addgene # 159538). Oligonucleotides with spacer sequences for target genes were inserted into the sgRNA cassette by ligation into the BspQI and BsmBI cloning sites.
Cloning of rAAV:HDR plasmids
Donor DNA fragments with target sites (rAAV:HDR:cleaved) or without target sites (rAAV:HDR:uncleaved) were synthesized as gBlocks (IDT) and cloned into the minimized Nme2Cas9+sgRNA AAV backbone plasmid following digestion of the latter with SalI restriction enzyme. The rAAV:HDR:uncleaved plasmids were cloned in NEB 5-alpha Competent E. coli cells (Catalog # C2987H), while rAAV:HDR:cleaved plasmids were cloned into the DH5α-AcrIIC4Hpa E. coli strain that we constructed. Oligonucleotides with spacer sequences for target genes were inserted into the sgRNA cassette by ligation into the BspQI cloning sites.
Creating E. coli DH5 cells stably expressing anti-CRISPR protein DH5α-AcrIIC4Hpa
Cells were created by λRed-promoted PCR-mediated recombination85. Briefly, acrIIc4Hpa, its promoter, and KanR cassette were cloned from plasmid pUC57mini-AcrIIC4Hpa (generously provided by Dr. Alan Davidson’s lab at the University of Toronto) into the pKIKOarsBKm (Addgene #46766) plasmid by restriction digestion and ligation into the arsB locus of DH5α. Cells harboring plasmid pKM208 (Addgene #13077) were cultured at 30 °C to an OD of 0.35, induced with 1 mM IPTG, and harvested at an OD of 0.80. Cells were prepared for electroporation (washing with 10% glycerol) and transformed with 500 ng of gel-purified PCR product (acrIIc4Hpa, its promoter, and KanR). After incubation at 37 °C overnight in kan-LB media, colonies were screened by PCR for the acr gene and the expected junction of the recombineering product with the arsB gene. Correct clones were passaged at 42 °C to eliminate the pKM208 plasmid.
The rAAV:HDR:cleaved plasmids were linearized with BspQI restriction enzyme and spacer sequences with compatible overhangs were ligated into 100 ng of plasmid backbone using ElectroLigase® (NEB M0369) following the manufacturer’s protocol. For electro-transformation, 2 μL of the ligation reaction was added to 40 μL of DH5α-AcrIIC4Hpa E. coli and electroporated at 2500 V, 200 Ω, 25 μF, in a 2 mm gap cuvette using Gene Pulser Xcell Electroporation Systems [Bio-Rad #1652660].
Vector production
Packaging of AAV vectors was done at the Viral Vector Core of the Horae Gene Therapy Center at the University of Massachusetts Medical School67. Constructs for HT-I studies were packaged in AAV8 capsids, whereas constructs for MPS-I studies were packaged in AAV9 capsids. A plasmid expressing anti-CRISPR protein (pEJS581; pCSDest2-AcrIIC4Hpa-FLAG-NLS; Addgene # 113436) was included in the triple-transfection packaging process to maintain intact rAAV:HDR:cleaved plasmids during production. Vector titers were determined by droplet digital PCR, and by gel electrophoresis followed by silver staining.
Cell culture
HEK293T cells harboring TLR-Multi-Cas-Variant 1 (TLR-MCV1)88, as well as Neuro2a cells (ATCC CCL-131), were cultured in Dulbecco’s Modified Eagle Media (DMEM, Thermo Fisher Scientific, Cat. No. 11965084); while MOLT-3 cells were cultured in RPMI-1640 medium. Both media were supplemented with 10% FBS (Thermo Fisher Scientific, Cat. No. 10438034) and 1% Penicillin/Streptomycin (GIBCO). Cells were incubated at a 37 °C incubator with 5% CO2.
Plasmid transfection
HEK293T cells and TLR-MCV1 cells were transfected using PolyFect Transfection Reagent (Qiagen, Cat No. 301105) with 400 ng of plasmid in a 24-well plate according to the manufacturer’s protocol. Neuro2a cells were transfected with 1000 ng of plasmid using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Cat. No. L3000015) following the manufacturer’s protocol. FACS analysis for the TLR-MCV1 cells were performed 3 days post-transfection using a Miltenyi MACSQuant VYB flow cytometer. HEK293T and Neuro2a cells were harvested 3 days post-transfection and genomic DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s protocol.
Ribonucleoprotein nucleofection
Nme2Cas9 protein was expressed in Rosetta (DE3) cells and purified using a Ni2+-NTA agarose column (QIAGEN)24. Imidazole was used to elute the bound protein followed by dialysis into storage buffer [20 mM HEPES-NaOH (pH 7.5), 1 mM DTT, 250 mM NaCl]. Wild-type (wt) gRNA was in vitro transcribed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) according to the manufacturer’s protocol. Truncated guide (Nme.sgRNA-100) was chemically modified with 2′-O-methyl analogs and 3′-phosphorothioates in 5′ and 3′ terminal RNA residues and was synthesized by Synthego (Redwood City, CA, USA). RNP complex was electroporated into HEK293T or MOLT-3 cells using the Neon transfection system. Briefly, 40 picomoles (HEK293T and MOLT-3 cells) of 3xNLS-Nme2Cas9 and 50 picomoles (HEK293T and MOLT-3 cells) of T7-transcribed or chemically synthesized sgRNA was assembled in buffer R along with 100,000 HEK293T cells or 200,000 MOLT-3 cells and electroporated using 10 μL tips. Electroporation parameters (voltage, width, number of pulses) were 1150 V, 20 ms, 2 pulses for HEK293T cells; 1600 V, 10 ms, 3 pulses for MOLT-3 cells.
TIDE analysis, amplicon sequencing, and indel analysis
PCR was carried out with TIDE or amplicon sequencing primers as shown in the Supplementary Table using High Fidelity 2x PCR Master Mix (New England Biolabs). PCR products were purified using the DNA Clean & Concentrator-100 (Zymo, Cat. No. D4029) and sent for Sanger sequencing using a TIDE forward primer (Supplementary Table). Indel readouts were obtained using the TIDE web tool (https://tide-calculator.nki.nl/)27. For amplicon sequencing, PCR was carried out using amplicon sequencing primers shown in the Supplementary Table. Libraries were sequenced for 600 cycles (300/300 PE) on a MiSeq (Fah locus), and for 300 cycles (SE) on a MiniSeq (Idua locus). FASTQ files were trimmed with Cutadapt (Galaxy Version 1.16.6)98 and analyzed using CRISPResso 2.0.40 with parameters specifying guide, amplicon, and expected HDR sequences. A no-guide sample was used as the negative control to determine the background. AAV integration was quantified using BWA-MEM (Galaxy Version 0.7.17.1) and samtools to align sequenced amplicons files to the Idua locus and AAV vector sequence99. We searched amplicon reads for HITI90 using CRISPResso 2.0.40, providing unedited, homology-repaired, sense HITI, and antisense HITI reference alleles.
Amplicon sequencing analysis of rAAV:HDR:cleaved religation after donor excision
We designed an NGS library using genomic DNA from liver tissues. Each primer was complementary to the U6 and U1a promoters flanking the donor (Supplementary Table). The gene-specific primer contained UMIs and adapter overhangs to multiplex the library. Samples were amplified by linear amplification (98 °C for 40 s, followed by 12 cycles of 98 °C for 45 s, 64 °C for 20 s, and 72 °C for 18 s) using the forward primers. A second PCR amplification was performed using a universal primer and a gene-specific reverse primer at 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s, 62 °C for 20 s, and 72 °C for 18 s, and extended at 72 °C for 5 m. PCR products were diluted (1:100) and amplified using Illumina barcoded primers at 98 °C for 30 s, followed by 30 cycles of 98 °C for 15 s, 65 °C for 20 s, and 72 °C for 75 s, and extended at 72 °C for 12 m. Equimolar amounts of the products were combined together and purified by gel extraction then AMPure PB beads.
We used CRISPResso 2.0.40 to align the reads to the intact AAV sequence, cleaved AAV sequence, and AAV sequence where the sequence between the cut sites is inverted. The aligned reads either mapped perfectly or contained small insertions or deletions as shown.
CRISPRseek off-target prediction and editing analysis
Bioconductor package CRISPRseek (Version 1.32.0) was used to predict off-target sites of Nme2Cas9 using mouse reference (mm10)24. The following parameters were sgRNA.size = 24, PAM = “NNNNCC,” PAM.size = 6, RNA.PAM.pattern = “NNNNCN,” and candidate off-target sites of a maximum of six mismatches. Top predicted off-targets with N4CC PAM and matching seed sequence to the on-target were analyzed by NGS using Genomic DNA liver tissue from mice. Illumina amplicon sequencing library was prepared using a two-step PCR protocol. Briefly, regions of predicted off-target loci (around 250 bp) were amplified from genomic DNAs using forward and reverse primers that contain Illumina adapter sequences (Supplementary Table). The PCR products were amplified using forward and reverse primers that contain unique barcode sequences. Products with the correct size were extracted from agarose gel and then purified by Ampure XP beads. The concentration of the final purified library was determined using Qubit (High Sensitivity DNA assay). The integrity of the library was confirmed by Agilent Tapestation using the Agilent High Sensitivity D1000 ScreenTape kit. The library was sequenced on an Illumina Miniseq platform according to the manufacturer’s instructions using Miniseq Mid Output Kit (300 cycles). Sequencing reads were demultiplexed on the Miniseq and CRISPResso 2.0.40 was used to align the reads and quantify editing efficiencies.
Vector library preparation and nanopore sequencing
Viral vector DNAs were extracted by phenol-chloroform extraction and ethanol precipitation66. Samples (<1 mg) were subjected to library preparations for Oxford Nanopore Technologies (ONT) sequencing, following the protocol for 1D Native barcoding of genomic DNA, using the Native Barcoding Expansion 1–12 (PCR-free) (EXP-NBD103) and Ligation Sequencing Kit (SQK-LSK108) components. Libraries were then multiplexed and sequenced on an ONT MinION instrument (Flow Cell R9.4.1, FLO-MIN106D).
Nanopore sequencing data analysis
MinKNOW software (v19.10.1) was used for base calling, adapter trimming, and demultiplexing. Sequencing reads were aligned to the appropriate reference sequences by BWA-MEM100 within the Galaxy web-based interface101,102,103,104. Alignments were visualized using Integrative Genome Viewer (IGV, version 2.6.3)105 with soft-clipping displayed. Start and end positions of aligned reads were defined with BEDTools (version 2.27.0)106,107. The abundances of start and end positions of all mapped reads were tabulated and plotted using GraphPad Prism 8.4.3.
SMRT sequencing
Amplicon libraries were constructed using locus-specific primers as shown in the Supplementary Table using Q5® High-Fidelity DNA Polymerase (New England Biolabs). One primer of each pair contains an 8-nucleotide UMI, and both contain adapter overhangs to multiplex the library using Bar. Univ. F/R Primers Plate-96v2 (Pacbio part no. 101-629-100). Forward and reverse primers were 1.1 and 1.9 kb from gRNA-I and gRNA-II Cas9 DSB sites, respectively. Each sample was amplified by linear amplification (98 °C for 40 s, followed by 10 cycles of 98 °C for 30 s, 64 °C for 20 s, and 72 °C for 75 s) using the forward primers. A second PCR amplification was performed using a universal PacBio primer and a gene-specific reverse primer at 98 °C for 30 s, followed by 30 cycles of 98 °C for 15 s, 64 °C for 20 s, and 72 °C for 75 s, and extended at 72 °C for 5 m. PCR products were diluted (1:100) and amplified using PacBio barcoded primers at 98 °C for 30 s, followed by 30 cycles of 98 °C for 15 s, 64 °C for 20 s, and 72 °C for 75 s, and extended at 72 °C for 12 m. Equimolar amounts of the products were combined together and purified by AMPure PB beads (PacBio, part no. 100-265-900) following the manufacturer’s recommendation. The libraries were sequenced on a Pacific Biosciences Sequel II Instrument in a 10-h collection SMRTCellTM. A circular consensus sequence (ccs) reads generated by SMRT Link (smrtlink/8.0.0.79519) using default parameters in fastq format were processed and analyzed using the Galaxy web platform at usegalaxy.org (20.09.rc1)108 with custom workflows, unless specified. Briefly, reads were demultiplexed using FASTQ/A Barcode Splitter (Galaxy Version 1.0.1) and converted to fasta to calculate and count read lengths within each library (Supplementary Fig. 2). To assign reads that represent segmental deletions, inversions, or indel events, reads were first re-IDed by appending each sequence identifier with the UMIs using fastp with (-U). Duplicated UMIs were removed from the analysis. Reads with mapping qualities >20 were then uniquely aligned using BWA-MEM 0.7.17 to reference sequences representing the wild-type loci and the predicted sequences following segmental deletion or inversion. The counts for reads aligning to the specified references were displayed as a percentage of all mapped reads. To categorize reads that mapped to the wild-type reference as either bearing indel events or unedited genomes at indicated target loci, reads were trimmed to ±100 nt surrounding each target PAM by Cutadapt (Galaxy Version 1.16.6). Trimmed reads were then converted to fasta and clustered by UCLUST (usearch v7.0.1090)109, a centroid-based clustering algorithm. Singlets were discarded and treated as reads bearing sequencing errors. Clustered reads using size reporting (-sizeout) were categorized into unedited and edited groups at each target site or both sites for dual-guide vectors and displayed as a percentage of all reads mapping to the respective wild-type sequence. To assess target loci that harbor vector genome integration events, fastq reads were mapped to the relevant vector genome reference and expressed as a percentage of all reads.
UDiTaS sequencing
Genomic DNA was quantified using the Qubit dsDNA BR kit according to the manufacturer’s instructions. About 200 ng of genomic DNA was tagmented with a Tn5 transposon loaded with custom oligos according to the protocol from ref. 63 (Supplementary Table). An initial round of nested PCR was done for one target with a gene-specific primer (Hpd_2.22_nest_FWD) paired with an i5-specific primer for ten cycles. This was followed by purification with Ampure beads at 0.9X and a second round with an interior gene-specific primer (Hpd_2.22_FWD or Hpd_4.10_REV) for 12 cycles. The PCR products were again purified with Ampure beads at 0.9X and a final PCR of 15 cycles was performed to add the Illumina i7 adapter and barcodes. Amplicons were double size-selected with Ampure beads at 0.5X/0.35X to yield a final size distribution of 200–1000 bp. Paired-end sequencing was performed on an Illumina Miniseq for 2 × 150 cycles.
Transposon-mediated target enrichment and sequencing at the Hpd locus after dual-guide editing was analyzed using the workflow outlined by Giannoukos et al.63 (UDiTaS v1.0) with modifications allowing for MiniSeq platform compatibility. Briefly, paired-end, dual-indexed libraries were demultiplexed using bcl2fastq (Illumina), masking UMIs (I2 nts 1–9). Next, un-demultiplexed UMIs were stored in a single FASTQ using bcl2fastq (–create-fastq-for-index-reads parameter), masking R1, I1, I2 nts 10–17, R2. Demultiplexed R1 and R2 FASTQs were paired with their corresponding UMIs using the fastq-pair110 hash lookup tool. Gzipped FASTQs were placed into individual UDiTaS-compatible sample directories (i.e., “parent/samplename/fastq_files/“). Per UDiTaS source code, FASTQs were named: samplename_R1.fastq.gz; samplename_R2.fastq.gz; samplename_umi.fastq.gz. Reference FASTA and 2-bit files were obtained from the UCSC table browser111. Zero-order primer and sgRNA coordinates were obtained from reference FASTA using the twobitreader python package. Sample metadata were saved as sample_info.csv in the “parent/” directory. Analysis was executed inside the umasstr/UDiTaS docker container, skipping demultiplexing.
RNA sequencing
Total RNA was isolated from liver tissues using TRIzol reagent and separated by chloroform. RNA-seq library preparation was performed with 1 ug of total RNA per sample using the TruSeq Stranded mRNA Library Prep kit (Illumina, 20020595) following the manufacturer’s protocol. Libraries were made for the following mice: three PBS-treated Fahneo/neo, eight dual-sgRNA Design 1 treated Fahneo/neo, eight dual-sgRNA Design 4 treated Fahneo/neo, three healthy C57BL/6, four dual-sgRNA Design 1 treated C57BL/6, four dual-sgRNA Design 4 treated C57BL/6, three PBS-treated FahPM/PM, three rAAV:FahDonor:ncSpacer treated FahPM/PM, three rAAV:ncDonor:FahSpacer treated FahPM/PM, ten rAAV:HDR:uncleaved treated FahPM/PM, and ten rAAV:HDR:cleaved treated FahPM/PM. Libraries were sequenced on an Illumina NextSeq 500 with single-end 75 nucleotide reads and an average of 19.88 million reads. All downstream analyses were performed with the Ensembl GRCm38.p6 (GCA_000001635.8) Mus musculus genome adapted to include the Nme2Cas9 mRNA sequence. Transcripts Per Million (TPM) were calculated with alignment-free quantification using Kallisto v0.45.0. Reads were mapped to the mm10 genome using STAR v2.7.5a in chimeric mode to identify chimeric junction reads and HTSeq v.0.10.0 was used to quantify read counts for each gene. Differential expression analyses were performed with DESeq2 release 3.11 after filtering out genes with less than ten read counts across all samples in the comparison. Gene ontology analyses were conducted with a custom script to test for enrichment of functional annotations among significantly differentially expressed genes, in order to avoid significant gene ontology terms with overlapping gene sets. Specifically, the script uses gene ontology annotation databases from the Gene Ontology Consortium to perform iterative enrichment analyses and p values are computed using a Fisher-exact test and then corrected using a Benjamini–Hochberg multiple test correction.
Gene expression heatmaps were created using the DolphinNext RNA-seq pipeline112 (revision 4), including Illumina adapter removal using the built-in trimmomatic software (v0.39). Default parameters were used to process raw reads using RSEM (v1.3.1) and STAR to map the reads to a reference mouse reference (mouse_mm10_refseq). Levels of differentially expressed genes were assessed using DESeq2 software (v1.28.1) using the following parameters: fit type: “parametric”, betraPrior = FALSE, test type = LRT” (Likelihood Ratio Test) and shrinkage = None. Alpha (padj) was set to 0.05 and a minimum fold change was set to 2. Heatmaps of (all) or (selected) up- and down-regulated genes were plotted with MRN normalization.
HPD western blotting
Liver tissue fractions were ground, resuspended in 200 μL of RIPA lysis buffer, and allowed to lyse for 15 m on ice. Measurements of total protein content was determined by Pierce™ BCA Protein Assay Kit (Thermo-Scientific) following the manufacturer’s protocol. A total of 25 μg of protein was loaded onto a 4–20% Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad) and transferred onto a PVDF membrane. After blocking with 5% Blocking-Grade Blocker solution (Bio-Rad) for 2 h at room temperature, membranes were incubated with rabbit anti-HPD (Sigma HPA038322, 1:600) overnight at 4 °C. Membranes were washed three times in TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Bio-Rad 1,706,515, 1:10,000) secondary antibodies for 2 h at room temperature. The membranes were washed four times in TBST and visualized with Clarity™ western ECL substrate (Bio-Rad) using a Bio-Rad ChemiDoc MP imaging system. Membranes were stripped using Restore™ Western Blot Stripping Buffer (Cat. number: 21059) following the manufacturer’s protocol and blocked with 5% Blocking-Grade Blocker solution (Bio-Rad) for 2 h at room temperature. The membranes were incubated with rabbit anti-GAPDH (Abcam ab9485, 1:2,000) overnight at 4 °C, washed three times in TBST, and incubated with HRP-conjugated goat anti-rabbit (1:4,000) secondary antibodies for 2 h at room temperature. The membranes were washed four times in TBST and visualized with Clarity™ western ECL substrate (Bio-Rad) as above.
Histological analysis
Liver tissues were harvested, and sections were placed in biopsy cassettes and fixed in 10% buffered formalin overnight before changing into 70% ethanol solution. FAH immunohistochemistry (IHC) was performed using an anti-FAH antibody (Abcam, Cat. No. ab81087; dilution 1:400), whereas HPD IHC was performed using an anti-HPD antibody (Sigma HPA038322; dilution 1:30). IHC and hematoxylin and eosin (H&E) staining were performed by the Morphology Core at the University of Massachusetts Medical School following standard procedures.
Reverse transcription PCR
Total RNA was isolated from liver tissues using TRIzol reagent and separated by chloroform. Reverse transcription reactions were carried out using SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific; 18080051) following the manufacturer’s protocol. The complementary DNA was used to quantify Fah gene expression in a qPCR reaction using primers shown in the Supplementary Table using PowerUp SYBR Green Master Mix (Applied Biosystems, Cat. No. A25742). Data were collected using QuantStudio Design and Analysis Desktop Software V1.5.1 on a QuantStudio 3 Real-Time PCR System (Applied biosystems). All reactions were normalized to gapdh and relative quantification in gene expression was determined using the 2−ΔΔCt method on Microsoft Excel Version 1902 (Build 11328.20644). PCR products were resolved by agarose gel electrophoresis.
Quantitative PCR for rAAV copy number
Genomic DNA from mouse tissue was used to quantify rAAV copy number using primers shown in the Supplementary Table using PowerUp SYBR Green Master Mix (Applied Biosystems, Cat. No. A25742). Data were collected using QuantStudio Design and Analysis Desktop Software V1.5.1 on a QuantStudio 3 Real-Time PCR System (Applied biosystems). All reactions were normalized to gapdh control and relative quantification of rAAV copies was determined using the 2−ΔΔCt method relative to PBS-injected cohorts on Microsoft Excel Version 1902 (Build 11328.20644).
Serum aspartate transaminase (AST) and alanine transaminase (ALT) assays
Blood was collected from mice by cardiac puncture immediately before euthanasia, and sera were isolated using a serum separator (BD, Cat. No. 365967) and stored at −80 °C. AST and ALT levels were determined using Cobas Clinical Chemistry Analyzer at the MMPC-University of Massachusetts Medical School Analytical Core (RRID:SCR_015365). Two mice in the PBS- and rAAV:ncDonor:FahSpacer negative control HT-I cohorts were hemolyzed, so we were able to measure ALT/AST for only one mouse. In the rest of the groups, data were presented as mean values ± s.e.m. (n = 3–7 mice per group).
Iduronidase, β-d-glucuronidase, and β-d-hexosaminidase activity assays
The three assays were done similarly using the indicated substrates93,113,114. Tissues were homogenized in ice-cold T-PER protein extraction reagent (Thermo Fisher Scientific, Cat. No. 78510) with protease inhibitor (Roche, Cat. No. 4693159001) using TissueLyser II (Qiagen). Quantification of total protein concentration was assessed by the bicinchoninic acid (BCA) method (Pierce, Cat. No. 23225). No more than 80 μg of total protein was used in enzymatic reactions (100 μL of total reaction volume), which includes sodium formate buffer, pH 3.5 (130 mM), d-saccharic acid 1,4-lactone monohydrate (0.42 mg/mL, Sigma-Aldrich, Cat. No. S0375), and 4MU-iduronic acid (0.12 mM, Santa Cruz Biotechnology, Cat. No. sc-220961). The reaction was incubated at 37 °C for 24–48 h and quenched with glycine buffer (pH 10.8). The fluorescence of released 4MU (excitation wavelength: 365 nm; emission wavelength: 450 nm) was detected using a fluorescence plate reader (BioTek) and compared against a standard curve generated using 4MU (Sigma-Aldrich, Cat. No. M1381). The iduronidase specific activity was calculated as 4MU released (pmole) per milligram of total protein per hour. The β-d-glucuronidase assay was done using 4MU-β-d-glucuronide (1 mM, Sigma, Cat. No. M9130) substrate, while the β-d-hexosaminidase activity assay was done using 4MU-β-d-hexosaminide (4 mM, Sigma, Cat. No. M2133) substrate and incubation time was set to 30 min.
Glycosaminoglycan (GAG) assay
Tissues were homogenized in a mixture of chloroform and methanol (2:1) using TissueLyser II (Qiagen) and dried in a Vacufuge (Eppendorf) to remove fat. The dried and defatted tissue was weighed and digested using papain (Sigma-Aldrich, Cat. No. P3125) at 60 °C overnight. The supernatant was used in the Blyscan assay to quantify GAG content using chondroitin-4-sulfate as standard (Accurate Chemical, Cat. No. CLRB1000). Levels of GAG were calculated as GAGs (microgram) per milligram of dried, defatted tissue114.
LAMP-1 western blotting
Liver tissues were homogenized in ice-cold T-PER protein extraction reagent (Thermo Fisher Scientific, Cat. No. 78510) with protease inhibitor (Roche, Cat. No. 4693159001) using TissueLyser II (Qiagen). The supernatant was used to quantify total protein concentration using the bicinchoninic acid (BCA) method (Pierce, Cat. No. 23225) and boiled with 4x Laemmli Sample Buffer (Bio-Rad, Cat. No. 1610747) at 95 °C for 10 min. Primary antibody rat anti-LAMP-1(BD Pharmingen, RUO – 553792) (1:2000 dilution) and secondary antibody LI-COR IRDye 680RD Goat Anti-Rat IgG (H + L) (LI-COR Biosciences, 926–68076) (1:5000 dilution) were used in western blot. The membrane was scanned with a LI-COR scanner (Odyssey). Western blot quantification analysis was performed with Image Studio Lite (LI-COR).
Humoral α-Nme2Cas9 immune response
A 96-well plate (Corning) was coated with 0.5 μg of recombinant Nme2Cas9 protein suspended in a 1× coating buffer (Bethyl E107). The plate was incubated for 12 h at 4 °C with shaking, then washed three times using 1x Wash Buffer (Bethyl E106). The plate was blocked with a blocking buffer (Bethyl E104) for 2 h at room temperature. After washing three times, diluted sera (1:40 in dilution buffer consisting of 1 L of TBS, 10 g of BSA, and 5 mL of 10% Tween 20) from mice injected with rAAV:Nme2Cas9 were added to each well in duplicates and incubated at 4 °C for 6 h. The plate was washed three times, and a goat anti-mouse HRP-conjugated antibody (100 μL; 1:100,000 dilution; Bethyl A90-105P) was added to each well. The plates were incubated at room temperature for 1 h, then washed four times. To detect immune responses, the plate was developed by adding 100 μL of TMB One Component HRP Substrate (Bethyl E102). After incubating in the dark for 30 min at room temperature, Stop Solution (Bethyl E115; 100 μL per well) was directly added. Absorbance was measured at 450 nm using a BioTek Synergy HT microplate reader and collected using the Gen5 software.
Statistical analysis
All data are presented as mean values ± s.e.m. p values are calculated using Student’s t-test (two-sided) and one-way ANOVA using GraphPad Prism 8.4.3.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
