2. MATERIALS AND METHODS2.1. Vectors designTwo AAV vectors were constructed: Cas9 vector containing the liver‐specific promotor‐1 (LP1) promotor and Streptococcus pyrogens Cas9 (spCas9) sequence, and single‐guide RNA (sgRNA) vector‐containing U6 promotor, sgRNA scaffold, and green fluorescent protein (GFP) sequence. We used the Benchling software (www.benchling.com) to select sgRNAs targeting exonic regions of the Ldha gene. We cloned annealed oligonucleotides (Biosune, China) into the BbsI site of the sgRNA vector.2.2. Cell culture and sgRNA selectionThe Pheochromocytoma (PC12) cells (American Type Culture Collection ATCC, VA) were cultured in a commercial growth medium (RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin). Approximately 2 × 105 cells were cotransfected with 0.5 μg of Cas9 vectors and 0.5 μg of sgRNA vectors in each well of a 24‐well plate. The media were replaced every 24 hours, and the GFP cells were collected via flow cytometry (FACS Calibur, BD) 72 hours after transfection. Genomic DNA was isolated with an extraction kit (Tiangen, China). DNA fragments were amplified (Table S1) and sequenced at the company (Biosune, China).The single‐strand annealing (SSA) luciferase assay was also performed to test the activity of sgRNAs. 24 , 25 , 26 Briefly, we generated a luciferase reporter plasmid that contained a luciferase gene, which was interrupted by stop codons, a DNA fragment of the rat Ldha gene, and flanking complementary repeat sequences. Upon cleavage of the target site by Cas9 nucleases, the complementary repeats restored the expression of the luciferase gene. The reporter plasmid, the Cas9, and sgRNA vector were cotransfected into HEK293T cells (ATCC, VA) and cultured for 48 hours. We used a luciferase detection system (Promega, CA) to determine the luciferase activity.2.3. AAV8 production and administrationThe procedure of AAV production was described in our previous study. 18 Briefly, AAV Rep2‐Cap8 and an adeno helper plasmid were cotransfected with the Cas9 vector or sgRNA vector in HEK293T cells using polyethylenimine (No. 24765‐2, Polysciences, PA). Sixty hours later, the cells were lysed and digested with Benzonase (No. 70746‐3, Merck‐Novagen, Germany), followed by freeze‐thaw cycles. Then, an iodixanol gradient ultracentrifugation was carried out to purify the viral vectors. The viral vectors were then concentrated 10‐fold to ∼1 mL using 15 mL, 100 kDa molecular weight cut‐off filters (Merck Millipore, Germany). AAV titration was determined by quantitative PCR (qPCR) method using SYBR Premix Ex Taq (Yeason, China). Newborn (postnatal day 7) male PH1 pups were injected with both 1 × 1012 vector genome (vg) of AAV8‐spCas9 and 1 × 1012 vg of AAV8‐sgRNA‐EGFP via the tail at a final volume of 200 μL. Each control newborn rat was injected with 1 × 1012 vg of AAV8‐sgRNA‐EGFP or with PBS only.2.4. Animal experimentsAll animal experiments conformed to the regulations drafted by the Institutional Animal Care and Use Committee of the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (XHEC‐F‐2017‐NSFC‐001) and the Shanghai Laboratory Animal Commission, and was permitted by the East China Normal University Public Platform for innovation (011). All AgxtD205N rats were genotyped as described, 18 with age‐matched Sprague Dawley (SD) rats used as controls. Animals were kept in a pathogen‐free facility on a 12 hours/12 hours light/dark cycle with ad libitum access to standard chow and water.Partial hepatectomy was performed 3 weeks after treatment to detect LDH expression, liver injury, and deep sequencing; the surgical procedure was carried out as previously reported. 18 Rats were sacrificed 6 months after vector treatment, and liver, kidney, skeletal muscle, heart, spleen, brain, and lung tissues were harvested for analysis.To test whether nephrocalcinosis was prevented, water with a 0.5% ethylene glycol (EG) (v/v) was given to four 6‐month‐old rats in each group for 4 weeks, during which time the rats were weighed weekly. Urine was collected 6 days after the EG challenge, and the rats were euthanized at the end of the fourth week for tissue harvest.2.5. On‐target and off‐target mutagenesis analysesThe genomic DNA of rat tissues was extracted using the phenol‐chloroform method. As shown in Table S2, candidate off‐target sites were identified using the Benchling software (www.benchling.com). The genomic region was amplified and the amplicons were sent for deep sequencing analysis (Novogene, Beijing). To detect indels in Ldha, we also designed a pair of primers to amplify the relevant sequence and sent it for deep sequencing. All primers are listed in Table S1. Finally, the high‐throughput sequencing data were submitted to CRISPR software (http://www.rgenome.net/cas-analyzer/) to assess and characterize indels. The data were uploaded to the NCBI Sequence Read Archive (SRA) database with the accession number PRJNA646016.2.6. Real‐time (RT)‐qPCRRNA was isolated using RNAiso Plus (TaKaRa, Japan). Reverse transcription was carried out using PrimeScript RT Master Mix (RR036A, TaKaRa, Japan). RT‐qPCR was performed on a QuantStudio3 real‐time PCR system (Applied Biosystems, MA) to measure rat Ldha, spCas9, EGFP, and β‐actin levels. Primers are listed in Table S1.2.7. Western blot analysisWestern blot analyses were performed on rat liver lysates, as described previously. 18 LDH protein was probed with an anti‐LDH antibody (ab52488, Abcam, UK) diluted at 1:2000. Mouse anti‐β‐actin antibody diluted at 1:2000 (A5441, Sigma, Germany) was used to detect β‐actin. An Odyssey Infrared Imaging System (LI‐COR, NE) was used for imaging of the blots.2.8. Sample collections and measurementsBlood was collected using retro‐orbital puncture of four rats from each group and sent for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) detection in serum (Servicebio, China). Urine was collected over 24 hours using metabolic cages and acidified with hydrochloric acid before measurement. The oxalate concentrations were determined by ion‐exchange chromatography using a Dionex ICS‐5000 (Thermo Scientific, MA).2.9. HistopathologyFor frozen sections, rat liver tissues were embedded in OCT compound, and imaged using an inverted laser scanning microscope (TCS SP8, Leica, Germany). Liver and kidney samples were fixed with 4% paraformaldehyde overnight, embedded in paraffin, and sectioned at 4 μm thick. Hematoxylin and eosin (H&E) stainings were performed according to standard protocols. For IHC, 5% hydrogen peroxide was used to block endogenous peroxidase, followed by overnight incubation with anti‐LDH antibody (ab52488, Abcam) to analyze LDH expression, anti‐CD68 (Bio‐Rad, CA) to trace inflammation, or anti‐α‐SMA (Invitrogen, CA) to detect liver fibrosis at 1:500, 1:200, and 1:200 dilutions, respectively. The slides were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies at 1:5000 dilution for 1 hour. Detection of HRP was performed as described in the DAB detection kit (SK‐4100, Vector Labs, CA). To visualize CaOx deposition in sections of kidney samples, Pizzolato staining was carried out as described previously. 27
2.10. Metabolite quantification and metabolomicsA total of 40 mg of liver sample was harvested from each rat; 50 μL ultra‐pure water and 10 μL 20 μM internal standards were added to the sample for homogenization. Then 390 μL precooled methanol/acetonitrile (1:1, v/v) was added to liver homogenate samples or to 40 μL plasma samples extracted from rats, followed by vortex mixing. The supernatant was harvested after precipitating the protein by centrifugation at 14 000 RCF at 4°C for 20 minutes. Quintuplicate samples were collected and sent for analysis by metabolon‐associated energy metabolism (Applied Protein Technology, China).2.11. StatisticsData are presented as means ± SD. Unpaired t‐tests were used to compare two groups (two‐tailed). The significant P‐values were denoted on the graphs, and a P < .05 was considered to be statistically significant.
Article TitleKnockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1
The data that support the findings of this study are available from the corresponding author upon reasonable request.