Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Juan Carlos Izpisua Belmonte ( belmonte@salk.edu ).

The HEK293A cell line was purchased from Invitrogen (Carlsbad, CA) and maintained in DMEM medium containing 10% fetal bovine serum (FBS), 2 mM glutamine, 1% non-essential amino acids, and 1% penicillin–streptomycin. Neuro-2a (N2a) cells were originally from Sigma-Aldrich and cultured with the same medium. Cas9 mouse embryonic stem cell (Cas9 mESCs) lines were derived from blastocysts of homozygous Rosa26-Cas9 knock-in mice using previously described procedures (). Cells were then maintained in N2B27media on Matrigel (Cultrex) coated plates. Female Cas9 mESC cell line was used in this study. This cell line was authenticated via morphology, PCR based genotyping, and sequencing.

ICR, C57BL/6, Rosa26-Cas9 knockin (Gt(ROSA)26Sor tm1.1(CAG-Cas9∗, -EGFP)Fezh , Stock No. 024858), and Dmd mdx (C57BL/10ScSn-Dmd mdx/ J, Stock No. 001801) mice were purchased from the Jackson laboratory. The mice were housed in a 12 hr light/dark cycle (light between 06:00 and 18:00) in a temperature-controlled room (22 ± 1°C) with free access to water and food. All procedures were performed in accordance with protocols approved by the IACUC and Animal Resources Department of the Salk Institute for Biological Studies. The ages of mice are indicated in each figure legend or panel. Both female and male mice were used for behavioral experiments, no notable sex-dependent differences were found in our analyses. For beta-cell ablation experiments, male mice were randomly assigned to experimental and control groups.

All animal procedures were performed according to NIH guidelines and approved by the Committee on Animal Care at the Salk Institute.

Method Details

Plasmid Design and Construction Esvelt et al., 2013 Esvelt K.M.

Mali P.

Braff J.L.

Moosburner M.

Yaung S.J.

Church G.M. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Liao et al., 2015 Liao H.K.

Gu Y.

Diaz A.

Marlett J.

Takahashi Y.

Li M.

Suzuki K.

Xu R.

Hishida T.

Chang C.J.

et al. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Suzuki et al., 2016 Suzuki K.

Tsunekawa Y.

Hernandez-Benitez R.

Wu J.

Zhu J.

Kim E.J.

Hatanaka F.

Yamamoto M.

Araoka T.

Li Z.

et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Swiech et al., 2015 Swiech L.

Heidenreich M.

Banerjee A.

Habib N.

Li Y.

Trombetta J.

Sur M.

Zhang F. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. The luciferase reporter (tLuc) was constructed by replacing mCherry with Luciferase in the M-tdTom-SP-gT1 plasmid (Addgene 48677) () and then sub-cloning this construct into the AAV backbone construct, as AAV-tLuc. The AAV-tLuc-mCherry reporter was constructed by inserting a 2A-mCheery fragment into AAV-tLuc. The U6-dgRNA-CAG-MPH plasmid was constructed by combining U6-MS2gRNA from the plasmid sgRNA(MS2)_cloning_backbone (Addgene 61424) and the MPH transactivation domain from the plasmid lenti_MS2-P65-HSF1_Hygro (Addgene 61426) under the control of a CAG promoter. U6-dgRNA-CAG-MPH was further sub-cloned into the AAV backbone to make AAV-U6-dgRNA-CAG-MPH. Either 20 bp or 14 bp spacers of gRNAs ( Table S1 ) were inserted into the plasmids with gRNA backbones at either the BsmBI or SapI site. The mock-gRNA target sequence was synthesized as described (). To generate different MS2 fused transcriptional activator constructs, VP64 and Rta were amplified from the SP-dCas9-VPR plasmid (Addgene 63798), and P65 was amplified from the MS2-P65-HSF1_GFP plasmid (Addgene 61423), which were subsequently sub-cloned into a pCAG-containing plasmid under the order described in Figure S1 . AAV-nEF-Cas9 was described previously (). AAV-CMVc-Cas9 was constructed by replacing the Mecp2 promoter of PX551 () with a core CMV promoter. AAV-nEF-dCas9 was constructed by replacing the Cas9 of AAV-nEF-Cas9 with dCas9 coding sequence.

Transfection of In Vitro Cultured Cells Lipofectamine 2000 or 3000 (ThermoFisher) was used to transfect HEK293 cells, N2a, and Cas9 mESCs. Transfection complexes were prepared following manufacturer’s instructions.

Luciferase-Based Reporter Assay After harvesting luciferase-expressing cells by TrypLE Express (Life Technologies), suspended cells were transferred to 96-well plates and reagents from Dual-Glo Luciferase Assay System (Promega) were applied. The luminescent signal was quantified using a Synergy H1 Hybird Reader (BioTek) with triplicated wells per sample.

AAV and Lentivirus Production Liao et al., 2015 Liao H.K.

Gu Y.

Diaz A.

Marlett J.

Takahashi Y.

Li M.

Suzuki K.

Xu R.

Hishida T.

Chang C.J.

et al. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. AAV2/9 (AAV2 inverted terminal repeat (ITR) vectors pseudo-typed with AAV9 capsid) viral particles were generated by or following the procedures of the Gene Transfer Targeting and Therapeutics Core at the Salk Institute for Biological Studies. Lentiviral vectors were packed as described and the vesicular stomatitis virus Env glycoprotein (VSV-G) was used ().

In Vivo Muscle Electroporation Wild-type or Cas9-expressing mice were anaesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). A small portion of the quadriceps muscle was surgically exposed in the hind limb. Plasmid DNA mixture (25 μg of each plasmid in 50 μL TE) was injected into the muscle using a 29-gauge insulin syringe. One minute following plasmid DNA injection, a pair of electrodes was inserted into the muscle to a depth of 5 mm to encompass the DNA injection site. Muscle was electroporated using an Electro Square Porator T820 (BTX Harvard Apparatus). Electrical stimulation was delivered in twenty pulses at 100 V for 20 ms. After electroporation, the open sites were closed by stitches and the mice were allowed to recover from the anesthesia on a 37°C warm pad.

Intramuscular (IM) AAV Injection Newborn (P2.5) mice were used for intramuscular injections. The AAV mixtures (AAV9-dgRNA (1 × 1011 GC); AAV9-tLuc reporter (1 × 1010 GC)) were injected into the tibialis anterior (TA) and quadriceps femoris (QA) muscles under anesthesia. For 3-week-old mice, mice were anaesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). A small portion of the quadriceps muscle was surgically exposed in the hind limb. The AAVs were injected into the TA muscle and/or the QF muscle using a 33 Gauge Hamilton syringe. After AAV injection, the skin was closed by stitches, and mice recovered on a 37 °C warm pad.

Facial Vein AAV Injection Gombash Lampe et al., 2014 Gombash Lampe S.E.

Kaspar B.K.

Foust K.D. Intravenous injections in neonatal mice. 11 GC); AAV9-tLuc reporter (1 × 1010 GC)) were injected via temporal vein of the P0.5 mouse. Newborn (P0.5) mice were used for facial vein injection as described (). The AAV mixtures (AAV9-dgRNA (1 × 10GC); AAV9-tLuc reporter (1 × 10GC)) were injected via temporal vein of the P0.5 mouse.

Intra-Cerebral AAV Injection Kim et al., 2014 Kim J.Y.

Grunke S.D.

Levites Y.

Golde T.E.

Jankowsky J.L. Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. 10 GC); AAV9-tLuc reporter (1 × 1010 GC)) were injected intracranially into neonatal mice. Neonatal mice were used for intra-cerebral injections as described (). The AAV mixtures (AAV9-dgRNA (5 × 10GC); AAV9-tLuc reporter (1 × 10GC)) were injected intracranially into neonatal mice.

Tail Vein AAV Injection C57BL/6 mice and Cas9 mice (males and females, 8 to 12 weeks old) received tail vein injection of AAV (AAV9-dgRNA (3.5 × 1012 GC)). Liver tissues and serum samples were collected 13 days after tail vein injections. Collected liver samples were used for qRT-PCR or fixed in 4% Paraformaldehyde (PFA), and then embedded in OCT compound after a PBS wash and quickly frozen in ethanol. Cryostat sections (10 μm) were labeled for Insulin, HNF3B, PDX1, or SIX2.

Bioluminescence Imaging (BLI) Mice were examined at each time point after electroporation or AAV infection for BLI analysis using an IVIS Kinetic 2200 (Caliper Life sciences, now PerkinElmer). Mice were injected intraperitoneally with 150 mg/kg D-Luciferin (Syn Lab), anesthetized with isoflurane, and then images were captured within 10 min of D-luciferin injection.

Cisplatin-Induced Acute Kidney Injury Mouse Model Imberti et al., 2015 Imberti B.

Tomasoni S.

Ciampi O.

Pezzotta A.

Derosas M.

Xinaris C.

Rizzo P.

Papadimou E.

Novelli R.

Benigni A.

et al. Renal progenitors derived from human iPSCs engraft and restore function in a mouse model of acute kidney injury. Li et al., 2016 Li Z.

Araoka T.

Wu J.

Liao H.K.

Li M.

Lazo M.

Zhou B.

Sui Y.

Wu M.Z.

Tamura I.

et al. 3D culture supports long-term expansion of mouse and human nephrogenic progenitors. Cas9 mice (males and females, 8 to 12 weeks old) received an intraperitoneal injection of 15 mg/kg cisplatin (Tocris Bioscience, Ellisville, Missouri) 8 days after tail vein injection of AAV. Kidney tissues and blood serum samples were collected 4 days after cisplatin administration. Blood serum was assayed for blood urea nitrogen (BUN) and serum creatinine (S-Cre) levels using commercially available assays (QuantiChrom Urea Assay Kit and QuaintChrom Creatinine Assay Kit; BioAssay Systems, Hayward, CA) as renal function parameters. Collected kidney samples were fixed in 4% paraformaldehyde (PFA) and embedded in OCT compound (Sakura Tissue-Tek) after PBS wash and quickly frozen in ethanol. Cryostat sections (10 μm) were stained with either hematoxylin and eosin (H&E) or periodic acid-Schiff’s reagent (PAS). Tubular necrosis, urinary casts, tubular dilation, and tubular borders were assessed in non-overlapping fields (high power field) as described ().

Beta Cell Ablation Induction of diabetes by high dose streptozocin (STZ) treatment was performed in Cas9 male mice that were 2-4 months old. A single STZ dose (160 mg/kg) in 0.1 M sodium citrate buffer (pH 4.5) was injected intraperitoneally after the mice were fasted for 5 hr. Forty-eight hours later, the mice were randomly grouped for injection of AAV9 with dgMock or dgPdx1 through tail vein. The blood glucose levels were measured every other day with a OneTouch Ultra 2 glucometer (OneTouch) using blood from the tail vein. The mice were sacrificed at indicated times and livers were dissected and processed for histological analysis.

Immunohistochemistry Tissues were harvested after transcardial perfusion using ice-cold PBS, followed by ice-cold 4% paraformaldehyde in phosphate buffer for 15 min. Tissues were dissected out and postfixed in 4% paraformaldehyde overnight at 4°C and cryoprotected in 30% sucrose overnight at 4°C and embedded in OCT (Sakura Tissue-Tek) and frozen on dry ice. For muscle, after tissue dissection, muscle was frozen in isopentane in liquid nitrogen. Serial sections at 10 μm were made with a cryostat and collected on Superfrost Plus slides (Fisher Scientific) and stored at −80°C until use. Immunohistochemistry was performed as follows: sections were washed with PBS for 5 min 3 times, incubated with a blocking solution (PBS containing 2% donkey serum (or 5% BSA) and 0.3% Triton X-100) for 1 hr, incubated with primary antibodies diluted in the blocking solution overnight at 4°C, washed with PBST (0.2% Tween 20 in PBS) for 10 min 3 times, incubated with secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 546, or Alexa Fluor 647 (Thermo Fisher) for 1 hr at room temperature. After washing, the sections were mounted with mounting medium (DAPI Fluoromount-G, SouthernBiotech). For muscle staining, antigen retrieval process was carried out by heating the sections for 20 min at 70°C in HistoVT One solution (Nacalai tesque) and washed two times with PBS. The primary antibodies used in this study were anti-Laminin, 1:100 (L9393, Sigma); anti-Pdx1, 1:100 (ab47267, Abcam); anti-Insulin, 1:100 (ab7842, Abcam); anti-Six2, 1:200 (11562-1-AP, Proteintech); anti-Hnf-3β, 1:100 (sc-101060, Santa Cruz) and anti-Utrophin, 1:50 (sc-15377, Santa Cruz).

RNA Analysis Total RNA was extracted from cells and tissue samples using either TRIzol (Invitrogen) or RNeasy Kit (QIAGEN) followed by cDNA synthesis using iScript Reverse Transcription Supermix for RT-PCR (Bio-Rad). qPCR was performed using SsoAdvanced SYBR Green Supermix and analyzed using a CFX384 Real-Time system (Bio-Rad). All analyses were normalized based on amplification of human or mouse Gapdh. Primer sequences for qPCR are listed in Table S2

Enzyme-Linked Immunosorbent Assay (ELISA) Mouse sera was subjected to ELISA assay following the standard protocol (Mouse Klotho ELISA kit, CUSABIO; Mouse IL-10 ELISA kit, Affymetrix eBioscience; Mouse Insulin ELISA kit, ALPCO). ELISA assays were performed in duplicate at three separate times, and the data are expressed as mean ± SD.

Wire Hang Test A single 2-mm diameter wire from a metal hanger was used in this test. The vertical distance between the wire and fall point was set at 37 cm. The mouse was lifted by the tail and allowed to grasp the middle of a metal wire with its forepaws. The hanging latency was recorded until each mouse fell. Two measurements were taken per mouse. The longest hanging time was used for statistical analysis.

Grip Strength Test Fore and hind limb grip strengths were assessed using a grip strength meter (Chatillon Force Measurement Systems, Largo, FL). Mice were lifted by the tail and its forepaws and backpaws were each allowed to grasp onto the steel grid attached to the apparatus. The mouse was then gently pulled across the steel grid until its grip was released. Mice were tested 5 times and the three highest measured values were averaged to calculate grip strength.

Chromatin Immunoprecipitation (ChIP)- Quantitative PCR Hatanaka et al., 2010 Hatanaka F.

Matsubara C.

Myung J.

Yoritaka T.

Kamimura N.

Tsutsumi S.

Kanai A.

Suzuki Y.

Sassone-Corsi P.

Aburatani H.

et al. Genome-wide profiling of the core clock protein BMAL1 targets reveals a strict relationship with metabolism. ChIP procedures were modified from a previous report (). Tissues were fixed in PBS containing 0.5% formaldehyde for 15 min. Glycine was added to a final concentration of 0.125 M, and the incubation was continued for an additional 15 min. After washing the samples with ice-cold PBS, the samples were homogenized in 1 mL of ice-cold homogenize buffer (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40, and protease inhibitors cocktail) and centrifuged (18,000 × g, 4°C, 5 min). The pellets were suspended in nucleus lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS, protease inhibitors) and sonicated 15 times for 10 s each time at intervals of 50 s with a Sonic Dismembrator 550 (Fisher Scientific). The samples were centrifuged at 18,000 g at 4°C for 5 min. Supernatants were diluted 10-fold in ChIP dilution buffer (50 mM Tris-HCl [pH 8.0], 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate, protease inhibitor). Nonspecific background was removed by incubating samples with a fish sperm DNA/protein A-agarose slurry at 4°C for 2 hr with rotation. The samples were centrifuged at 1,000 g at 4°C for 2 min, and a 0.1 volume of the recovered supernatants was stored as an input sample, whereas the rest was incubated overnight with 2 μg of indicated antibodies at 4°C with rotation. The immunocomplexes were collected with 50 μL of a fish sperm DNA/protein A/G-agarose (sc-2003, Santa Cruz) at 4°C for 3 hr with rotation. The beads were sequentially washed with the following buffers: radioimmunoprecipitation assay (RIPA) buffer-150 mM NaCl, RIPA buffer-500 mM NaCl, and LiCl wash solution. Finally, the beads were washed twice with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The immunocomplexes were then eluted by the addition of 200 μL of ChIP direct elution buffer (10 mM Tris-HCl [pH 8.0], 300 mM NaCl, 5 mM EDTA, 0.5% SDS) and rotated for 15 min at room temperature and incubated for 4 hr at 65°C. The DNA was recovered by phenol-chloroform-isoamyl alcohol (25:24:1) extraction and ethanol precipitation. H3K4me3 (ab8580, Abcam), H3K27ac (MA309B, Takara), and IgG-bound DNA were used for quantitative real-time PCR (qRT-PCR). The primers were designed as Table S3

Surveyor Assay Sanjana et al., 2012 Sanjana N.E.

Cong L.

Zhou Y.

Cunniff M.M.

Feng G.

Zhang F. A transcription activator-like effector toolbox for genome engineering. The indel frequency was analyzed by surveyor assay (IDT). Briefly, samples were collected to extract genomic DNA by DNeasy Blood & Tissue kit (QIAGEN). The Il-10 or Pdx1 locus was amplified by PCR from 100 ng of genomic DNA using LA Taq Hot Start polymerase (TaKaRa) and Il-10 primers (forward: 5′- ccagttctttagcgcttacaatgc-3′ and reverse: 5′-gcagctctaggagcatgtgg-3′) or Pdx1 primers (forward: 5′-aagctcattgggagcggttttg-3′ and reverse: 5′-gtccggaggacttccctgc-3′) in a 20 μL reaction. PCR product (200 ng) was then denatured and slowly re-annealed using a stepwise gradient temperature program in a T100 thermocyler (Bio-Rad), followed the protocol adapted from previous publications ().

DNA Library Preparation and Deep Sequencing Il-10 primers for the Surveyor assay were used for the first round of amplifications in the nested-PCR procedure with limited PCR cycles using 100ng of genomic DNA from cultured cells or tissues. This PCR product was used for the second round of amplification in the nested-PCR procedure using primer pairs with deep sequencing adaptor (mIl10-adaptor-F1: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTcatggtttagaagagggagga-3′ and mIl10-adaptor-R1: 5′-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTgagcaggcagcatagcagt-3′). The nested PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN) for DNA library preparation. NEB NextUltra DNA Library Preparation kit was used to prepare the sequencing library (Illumina, San Diego, CA, USA). Adaptor-ligated DNA was indexed and enriched by limited cycle PCR. The DNA library was validated using TapeStation (Agilent Technologies, Palo Alto, CA, USA), and was quantified using a Qubit 2.0 Fluorometer. The DNA library was quantified by real time PCR (Applied Biosystems, Carlsbad, CA, USA). The DNA library was loaded onto an Illumina MiSeq instrument (Illumina, San Diego, CA, USA). Sequencing was performed using a 2x150 paired-end (PE) configuration by GENEWIZ, Inc. (South Plainfield, NJ, USA). The MiSeq Control Software (MCS) on the MiSeq instrument conducted image analysis and base calling. The raw sequencing reads were quality and adaptor trimmed using Trimmomatic-0.36. The reads were aligned to the target gene reference genome using bwa-0.7.12. The variants were called for each sample using mpileup within samtools-1.3.1 followed by VarScan-2.3.9. At least 50,000 reads per sample was analyzed and the variant frequency for the indel was set above 0.25% of total reads to compare with the region of gRNA targets.