The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

Here, we describe an LNP-based delivery method for in vivo editing with CRISPR/Cas9 components that result in >97% knockdown of the mouse TTR protein. This system uses a biodegradable lipid to form LNPs co-formulated with both Spy Cas9 mRNA and sgRNA. Notably, we provide evidence of LNP mediated, in vivo, CRIPSR/Cas9 genome editing at clinically relevant levels sustainable over a 52-week period following a single dose. Our studies have identified a chemical modification pattern in sgRNA that achieves high levels of durable in vivo editing. The method described here produces robust and durable knockdown in a single administration, suggesting that this system offers a clinically viable method for the therapeutic delivery of CRISPR/Cas9-based gene editing in the liver.

Unlike viral delivery systems, lipid nanoparticles (LNPs) meet all of these criteria. They have been extensively validated both pre-clinically and clinically for the delivery of small interfering RNA (siRNA) ( ClinicalTrials.gov identifier: NCT01960348 ) and mRNA (). Recent reports have shown some success in using LNPs to deliver CRISPR/Cas9 components, but editing efficiencies have fallen short of clinically relevant levels ().

The ideal delivery system for CRISPR/Cas9 requires a number of key attributes that include (1) a transient, non-integrating Cas9 expression construct to limit potential off-target events, immune responses, and integration events into the genome; (2) efficient delivery with the capacity to transport the large Cas9 enzyme (or its encoding mRNA) as well as one or more sgRNAs; and (3) the option to redose should the effect wane over time or require repeat dosing to reach a therapeutically relevant level of editing; and (4) scalability of the formulation to enable large-scale manufacturing to address both orphan and common diseases (e.g., TTR amyloidosis [ATTR] and hepatitis B virus [HBV], respectively).

Current delivery methods build on the protocols that were developed for the delivery of traditional gene therapy and have been optimized over the past few decades. Viral vectors are the most common delivery method for introducing CRISPR/Cas9 components into pre-clinical models of disease (). However, these vectors suffer from potential limitations: they are capable of triggering both pre-existing and adaptive immune responses () and expression from these vectors is typically sustained, adding unnecessary risks of immunogenicity and potential side effects.

Genetic engineering has enabled breakthroughs in biomedical research, and as technology has evolved, it has accelerated the pace of the discovery of exciting new industrial and medical applications. The latest step in this evolution is CRISPR/Cas9 genome editing technology. Originally identified as a bacterial immune system to protect against bacteriophage infection, the engineered CRISPR/Cas9 system can precisely edit and modify any location in the genome (). CRISPR/Cas9 holds tremendous promise for the treatment of human disease, with the ultimate potential to cure genetic diseases through (1) correcting disease-causing or associated mutations in the genome, (2) inactivating deleterious or aberrant protein expression, or (3) inserting therapeutically relevant DNA in a targeted fashion. However, these applications face technical hurdles as they are brought to patients, with challenges including safe and effective in vivo delivery of CRISPR/Cas9 components.

In conclusion, our results demonstrate a non-viral delivery approach that can achieve high levels of durable in vivo CRISPR/Cas9-mediated gene editing, offering therapeutically meaningful levels of editing in a single, systemic administration. With an optimized guide sequence, a specific sgRNA chemical modification pattern, the ability to re-administer, and a biodegradable ionizable lipid, these LNPs meet all of the criteria essential for CRISPR/Cas9 delivery system: they are completely synthetic and scalable, are non-viral, and have the potential to serve as a clinically viable treatment for liver-based genetic diseases.

In these LNP formulations, the majority of the cargo accumulates in the liver; however, we do observe low, but detectable, levels of editing in other tissue including the spleen and kidney ( Figure 4 C). It is anticipated that editing of a hepatocyte-specific expressed gene in non-hepatic tissue should have limited or no safety implications because knockout or repair of deleterious mutations in an off-target tissue should not have any functional consequence.

Durability is an essential measure of the effectiveness of any genome editing approach. The cellular source of new hepatocytes during homeostasis or liver injury has been extensively investigated over the past decades, with evidence supporting both a hepatocyte-to-hepatocyte regeneration model as well as a dedicated stem cell model () depending on the status of the liver and methods used. A recent paper provides evidence for a self-renewing pericentral hepatocyte population that is responsible for homeostatic renewal of the liver (). These WNT-responsive, AXIN2+, glutamine synthetase (GS)+ diploid cells surround the central vein, are able to self-renew, and can replace ∼40% of hepatocytes in the mouse liver in 1 year. Delivery of GFP mRNA using LNP-INT01 demonstrates efficient delivery to the majority of hepatocytes in the mouse liver, including GS+ pericentral cells ( Figure S4 B). Interestingly, even at sub-maximal levels (0.3 and 1 mpk groups), editing is consistent over a 12-month period. This could imply that the ratio of edited cells in the stem cell population is similar to the bulk hepatocyte population, or that the majority of hepatocyte turnover is from existing hepatocytes. Regardless of the mechanism of hepatocyte turnover/regeneration, given that LNP mediated editing is remarkably stable even after 1 year, it is clear that editing is occurring in the cell population responsible for long term homeostatic repopulation of the liver. With editing and protein knockdown consistent for at least twelve months, LNP-INT01 produces the most durable in vivo editing demonstrated to date.

LNP-INT01 provides the most effective in vivo delivery of CRISPR/Cas9 components reported to date. A recent study has shown some success in using non-viral delivery of Cas9 mRNA in combination with viral vectors to deliver guide RNA; however, editing efficiencies were modest (∼25%), and immune responses remain a significant risk with this strategy (). Other groups have reported in vivo editing using non-viral delivery systems, with efficiencies ranging from ∼3.5% of hepatocytes () to ∼35% editing in the liver after 4 systemic doses (2 with Cas9 mRNA LNP, 2 sgRNA LNP) (). In contrast, LNP-INT01 combined with a sgRNA having a specific chemical modification pattern yields more than 70% editing and >90% knockdown with a single dose of the co-formulated LNP.

Our data suggest that LNP-INT01 is a robust and effective delivery system for CRISPR/Cas9 in mice; however, previous studies have shown that LNPs can behave differently in other species. Therefore, we tested our LNP-INT01 delivery system in rats as an additional validation of the efficacy and clinical relevance of the method in a larger animal model. Three different rat Ttr-specific guides were co-formulated with Cas9 mRNA and were systemically dosed at 1, 2 or 5 mpk. Dose-dependent editing was observed with all three guides: editing in the liver reached nearly ∼70%, while serum levels of TTR were reduced more than 90% ( Figure 4 ). These results demonstrate that LNP-INT01 is also effective in rats, an important preclinical species that is significantly (∼10 fold) larger than mice.

(A and B) Sprague Dawley rats (n = 5/group) were systemically dosed with LNPs co-formulated with Cas9 mRNA and highly modified rat-specific Ttr guides (G531, G533, and G534) at 1, 2 or 5 mpk or PBS (con). Rats were sacrificed 9 days post-administration, and liver editing (A) and systemic TTR levels were measured (B). Each data point indicates one animal with the mean and SEM indicated.

In contrast with viral delivery systems, the synthetic nature of LNPs may allow for multi-dosing regimens, something that has been demonstrated in the clinic for LNP-based siRNA delivery systems ( ClinicalTrials.gov identifier: NCT01960348 ). To investigate whether multi-dosing is possible with LNP-INT01, mice were administered weekly (4) or monthly (3) doses of LNP-INT01 at 0.5 mpk or a single 2 mpk dose. Results of weekly or monthly dosing schedules demonstrate cumulative editing after multiple administrations ( Figure 3 ), permitting the use of multiple doses to reach a desired editing level, or longer term maintenance of editing levels.

(A and B) CD-1 mice (n = 5/group) were systemically dosed with LNPs co-formulated with Cas9 mRNA and a highly modified murine Ttr-specific guide either weekly (A) or monthly (B). In the weekly dosing group, mice were dosed with 0.5 mpk on a weekly basis and were sacrificed 1 week after dosing, while the monthly group was dosed monthly and also sacrificed 1 week after dosing. As a positive control, mice were dosed with a single 2-mpk dose (black bar). Liver editing was measured by NGS, and mean + SEM are shown.

As Ttr is expressed primarily in hepatocytes, and these cells account for ∼50%–60% of the total cells in the liver (), these data suggest that the percentage of editing in the hepatocyte population is likely higher than that observed in the total liver. The heterogenous, polyploid nature of hepatocytes makes determining the percentage of hepatocyte-specific alleles in a given liver biopsy challenging, but sequencing of hepatocytes isolated from LNP-treated mice yielded higher editing percentages than liver biopsies ( Figure S4 A). This high level of protein knockdown suggests that editing is highly efficient, occurring at nearly all loci that express Ttr.

Given the rapid turnover of the LNP components, we next assessed the durability of editing and knockdown in the liver. LNP-INT01, co-formulated with both Cas9 mRNA and highly modified sgRNA, was injected into CD-1 mice at 0.3, 1, or 3 mpk (mg/kg), and mice were followed for 1 year. Importantly, we found that LNP-INT01 administration resulted in highly durable effects, with a dose-dependent increase in liver editing and decrease in serum TTR levels remaining stable for at least 12 months ( Figures 2 F and 2G). At the highest dose, we observed >97% knockdown of serum TTR levels, with a corresponding ∼70% editing of DNA across the liver. Immunohistochemistry (IHC) analysis of liver sections confirmed the long-term, dose-dependent knockdown of TTR ( Figures 2 H and S3 ).

The majority of LNPs currently used in clinical applications contain a non-degradable ionizable lipid (), resulting in bioaccumulation in the liver and potential safety risks. To address this issue, LP01 was designed to be biodegradable via labile ester linkages. LP01 from LNP-INT01 was cleared from the liver with a Tof ∼6 hr ( Figure 2 C). This degradation is in contrast with the stability of DLin-MC3-DMA, a lipid currently being used in clinical trials, which shows no appreciable degradation over 24 hr (). LNP-INT01 administration was safe and well tolerated in both mice and rats with respect to cytokine stimulation and body weight loss ( Figures S2 C–S2H).

A main disadvantage of most viral delivery systems is the sustained expression of Cas9 and the guide RNA. To assess the pharmacokinetics of LNP-INT01, we quantified the amount of Cas9 mRNA and end-modified sgRNA remaining in both the plasma and liver after injection ( Figures 2 A and 2B , respectively). We found that both molecules were rapidly cleared from the animals and were undetectable after 72 hr. Cas9 protein levels appear to peak at ∼4 hr post-administration ( Figure S2 B). Despite the transient nature of the CRISPR components, the level of editing in the liver increased over time, reaching maximal levels after 96 hr ( Figures 2 D and 2E). Possible explanations for the discrepancy between cargo clearance time and editing maximum include the stability of the RNP complex after cargo components have degraded, the time required for repair following induction of a double-stranded break (), and/or that a small percentage of cargo released into the cytoplasm (<2%–3%) () may have a longer half-life than did the cargo directly shuttled into lysosomes.

(A and B) Mice were sacrificed (n = 3) at the indicated time points, and Cas9 mRNA and sgRNA were quantified in plasma (A) and in liver (B) (mean and SD are shown). Both Cas9 mRNA and sgRNA showed similar half-lives in plasma (T1/2 = 2.32 hr [sgRNA] vs. 2.54 hr [mRNA]) and in liver (T1/2 = 2.43 hr [sgRNA] vs. 2.09 hr [mRNA]).

While these levels of editing were significant, we sought additional approaches, including further chemical modification of the guide RNA, to increase LNP potency. We first performed an in vitro screen using dgRNA to identify regions of the guide that were amenable to chemical modification. Based on these data, sgRNAs with the most promising modifications were evaluated in vivo ( Figure 2 A). We identified a modification pattern ( Figure 1 D) that increased in vivo activity significantly compared to unmodified or end-modified guides ( Figures 1 E and 1F). The increased activity was independent of the guide target sequence, as the improved activity was observed across multiple Ttr target sequences. Unmodified guides had minimal in vivo activity, highlighting the importance of sgRNA chemical modification for robust in vivo activity. One hypothesis for the increased activity is that chemical modifications stabilize the sgRNA, protecting it from degradation until sufficient Cas9 protein has been translated and is available for ribonuclear protein (RNP) formation.

Our preliminary studies showed that formulation of Cas9 mRNA with single guide RNA (sgRNA) was superior to dual-guide RNA (dgRNA) and that co-formulation was more efficacious than split formulation ( Figures S1 E and S1F). All formulations had high encapsulation efficiencies (>95%) and were less than 100 nm in size ( Table S1 ). Based on this initial work, we screened a series of Ttr-specific guides in vivo ( Figure 1 A). End-modified sgRNAs were co-formulated with Cas9 mRNA into LNP-INT01, and mice were harvested 7 days post-systemic administration for analysis. Compared to PBS-treated mice (<0.2% editing), a range of editing efficiencies were observed across the different guide sequences ( Figure 1 A). Serum TTR level and editing efficiency were well correlated (R= 0.67), with an even stronger correlation observed when more than 20% of the liver cells were edited (R= 0.787) ( Figures 1 B, S1 C, and S1D). When these LNPs were assayed for in vitro activity, a strong correlation between in vivo and in vitro activity was observed ( Figure 1 C).

(E and F) LNPs co-formulated with Cas9 mRNA and two different sgRNAs (G284, E; G269, F) targeting Ttr were administered to CD1 mice at 0.3, 1, or 2 mpk (n = 5 per group; mean and SD shown). 7 days later, liver editing was measured by NGS. Mean and SD are shown.

Lipid-based formulations have demonstrated the ability to deliver certain cargos to a range of tissue and cell types (), and, upon systemic administration, are known to interact with serum proteins, including apolipoprotein E (ApoE), facilitating efficient uptake by hepatocytes in a receptor-mediated manner (). This makes them useful for targeting endogenous genetic defects in liver hepatocytes. Indeed, LNP-INT01 was able to efficiently deliver GFP mRNA to the mouse liver following systemic administration ( Figure S1 B). To test the efficacy of LNP-INT01 to deliver CRISPR/Cas9 components, we designed guide RNAs for the mouse transthyretin (Ttr) gene, the homolog of a target for therapeutic gene editing to treat amyloidosis in humans.

We have developed an LNP-based delivery system, called “LNP-INT01,” that allows Cas9 mRNA and guide RNA to be co-formulated into a single particle for simultaneous delivery in a single dose. LNP-INT01 comprises a biodegradable, ionizable lipid—termed “LP01” ( Figure S1 A), with an approximate pKa of 6.1—helper lipid, and PEG-DMG.

Experimental Procedures

LNP Formulation Leung et al., 2012 Leung A.K.

Hafez I.M.

Baoukina S.

Belliveau N.M.

Zhigaltsev I.V.

Afshinmanesh E.

Tieleman D.P.

Hansen C.L.

Hope M.J.

Cullis P.R. Lipid nanoparticles containing siRNA synthesized by microfluidic mixing exhibit an electron-dense nanostructured core. LNPs were formulated with an amine-to-RNA-phosphate (N:P) ratio of 4.5. The lipid nanoparticle components were dissolved in 100% ethanol with the following molar ratios: 45 mol% LP01 lipid, 44 mol% cholesterol, 9 mol% DSPC, and 2 mol% PEG2k-DMG. The RNA cargo (1:1 weight ratio mRNA:sgRNA) was dissolved in 50 mM acetate buffer (pH 4.5), resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr Benchtop Instrument, in accordance with the manufacturer’s protocol. After mixing, the LNPs were collected and diluted in PBS (approximately 1:1), and then the remaining buffer was exchanged into PBS (100-fold excess of sample volume) overnight at 4°C under gentle stirring using a 10 kDa Slide-a-Lyzer G2 Dialysis Cassette (ThermoFisher Scientific). The resultant mixture was then filtered using a 0.2-μm sterile filter. The filtrate was stored at 2°C–8°C. Note that the multi-dose and rat formulations ( Figures 3 and 4 ) were formulated using 25 mM citrate, 100 mM NaCl cargo buffer (pH 5), and buffer exchanged by TFF into tris-saline sucrose buffer (TSS) buffer (5% sucrose, 45 mM NaCl, and 50 mM Tris) and had an average size of 105 nm ( Table S1 ). Encapsulation efficiencies were determined by ribogreen assay (). Particle size and polydispersity were measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. Representative DLS spectra can be seen in Figures S4 D and S4E.

RNA Production Capped and polyadenylated Cas9 mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The Cas9 open reading frame (ORF) contained 2 C terminal nuclear localization sequence (NLS) and an HA tag. The Cas9 mRNA was purified from enzyme and nucleotides using a MegaClear Transcription Clean-up Kit, in accordance with the manufacturer’s protocol (ThermoFisher). The transcript concentration was determined by measuring the light absorbance at 260 nm (Nanodrop), and the transcript was analyzed by capillary electrophoresis by Bioanalyzer (Agilent). Hendel et al., 2015 Hendel A.

Bak R.O.

Clark J.T.

Kennedy A.B.

Ryan D.E.

Roy S.

Steinfeld I.

Lunstad B.D.

Kaiser R.J.

Wilkens A.B.

et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Chemically synthesized sgRNA was obtained from a commercial supplier (AxoLabs) with the appropriate chemical modification pattern. The end modified pattern is as previously described (). The use of the term highly modified guide, or HM, in this paper specifically refers to the chemical modification pattern described in Figure 1 D.

Editing Quantification PCR primers were designed around the target site, and the genomic area of interest was amplified. Additional PCR was performed in accordance with the manufacturer’s protocols (Illumina) to add the necessary chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the mouse reference genome after eliminating those having low quality scores. The resultant files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected, and the number of wild-type reads versus the number of reads that contain an insertion, substitution, or deletion was calculated. The editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of sequence reads with insertions or deletions over the total number of sequence reads, including wild-type.

LNP Delivery In Vitro Mouse primary hepatocytes (Invitrogen) were cultured at 15,000 cells per well in hepatocyte plating media (Invitrogen) using collagen-coated 96-well plates. After 5 hr, the plating media was removed and replaced with hepatocyte maintenance media containing LNPs and 3% mouse serum (pre-incubated for 5 min at 37°C). Cells were transfected for 42–48 hr prior to lysis and next-generation sequencing (NGS) analysis. LNPs were diluted and added to cells starting at 100 ng Cas9 mRNA and approximately 30 nM guide RNA per well, carrying out serial dilutions in a semi-log manner down to 0.1 ng Cas9 mRNA and 0.03 nM guide RNA per well.

LNP Delivery In Vivo All procedures for animal experimentation were approved by the Intellia Institutional Animal Care and Use Committee and conducted in accordance with their guidelines. CD-1 were used for these studies because they are routinely used for toxicity studies, allowing for pharmacokinetic/pharmacodynamic modeling (PK/PD) and toxicity studies in the same mouse strain. Female mice ranging from 6–10 weeks of age were used in each study. LNPs were dosed via the lateral tail vein in a volume of 0.2 mL per animal. Excipient-treated animals were used as negative controls for all studies. Animals were euthanized at various time points by exsanguination via cardiac puncture under isoflurane anesthesia. For studies involving in vivo editing, liver tissue was collected from the median or left lateral lobe from each animal for DNA extraction and analysis. Female Sprague-Dawley rats ranging from 6–8 weeks of age were used. Formulated material was dosed at 5, 2, and 1 mg/kg in a dose volume of 3 mL/kg body weight. Animals were euthanized by exsanguination under isoflurane anesthesia. Blood was collected into serum separator tubes for circulating TTR quantitation by ELISA, and a small biopsy from the median lobe of the liver was collected for DNA extraction for NGS.

Cytokine Analysis 50–100 μL of blood was collected by tail-vein nick for serum cytokine measurements, usually 4 hr post-administration. Blood was allowed to clot at room temperature for approximately 2 hr and then centrifuged at 1,000 x g for 10 min before collecting the serum. A Luminex-based magnetic bead multiplex assay (mouse cytokines: Affymetrix ProcartaPlus, catalog number Exp040-00000-801; rat: Milliplex Map Kit, cat# RECYMAG65K27PMX) was used for cytokine analysis on collected samples. Serum was diluted 4-fold using the sample diluent provided, and 50 μL was added to wells containing 50 μL of the diluted antibody coated magnetic beads. The plate was incubated for 2 hr at room temperature and then washed. Diluted biotin antibody (50 μL) was added to the beads and incubated for 1 hr at room temperature. The beads were washed again before adding 50 μL of diluted streptavidin PE to each well, followed by incubation for 30 mins. The beads were washed once again, then suspended in 100 μL of wash buffer, and read on the Bio-Plex 200 instrument (Bio-Rad). The data were analyzed using a Bioplex Manager (v.6.1) analysis package with cytokine concentrations calculated from a standard curve using a five parameter logistic curve fit.

TTR ELISA Analysis Total TTR serum levels were determined using a Mouse Pre-albumin (Transthyretin) ELISA Kit (Aviva Systems Biology, cat. OKIA00111) or Rat Pre-albumin ELISA Kit (Aviva Systems Biology, cat. OK1A00159). Serum was diluted to a final dilution of 10,000-fold with 1× assay diluent. The plate was read on a SpectraMax M5 plate reader at an absorbance of 450 nm. Serum TTR levels were calculated by SoftMax Pro software (v.6.4.2) using a four parameter logistic curve fit from the standard curve. Final serum values were adjusted for assay dilution.

LP01 Liquid Chromatography-Tandem Mass Spectrometry Quantification Blood samples for the quantitation of LP01 were drawn into K2EDTA tubes, processed to plasma, and snap frozen in liquid nitrogen. Liver samples were taken after cardiac puncture and perfusion of the liver until blanched. Segments of about 100 mg were resected and snap frozen in liquid nitrogen. All samples were stored at −80°C until analysis. Homogenization buffer was added to tissue samples, and ceramic beads (Matrix D) were used to homogenize the tissue in a bead mill (FastPrep, MP Biomedicals, Santa Ana, CA, USA). Aliquots of the supernatant and plasma were mixed with 1:1 acetonitrile:isopropanol to induce protein precipitation. An internal standard (d10-LP01) was added to all samples. After centrifugation (5 min at 1,300 × g) the supernatants were transferred to a 96-well plate. A standard curve LP01 was created covering the concentration range of 1–10,000 ng/ml. Samples expected to exceed these concentrations were diluted accordingly. Concentrations were determined by multiple reaction monitoring using an high-performance liquid chromatography (HPLC) triple-quadrupole mass spectrometer (Agilent 1200, Santa Clara, CA, USA; API5500, Sciex, Framingham, MA, USA). Plasma and tissue concentrations were determined from the standard curve using Analyst 1.6.2 (Sciex, Framingham, MA, USA).

Hepatocyte Isolation Mice were dosed with LNP containing Cas9 mRNA and single guide targeting TTR. At 7–18 days after the dose, livers were perfused, first with HBSS/0.5 mM EDTA and then with DMEM containing 0.8 mg/ml type 1 collagenase. Hepatocytes were filtered through a 70-μm cell strainer and allowed to adhere to a gelatin-coated cell culture dish for 3 hr. Cells were lysed for RNA isolation using the RNeasy Mini Kit (QIAgen) or for DNA isolation using the PureLink Genomic DNA Mini Kit (Invitrogen).

GFP and GS IHC/Immunofluorescence Liver samples were fixed in 10% buffered formalin for 24 hr, followed by 70% ethanol. Tissue were embedded in paraffin and sectioned. Antigen retrieval was performed in sodium citrate, and IHC for GFP was performed with rabbit polyclonal anti-GFP (Novus Biologic) and detected either with a goat anti-rabbit horseradish peroxidase (HRP) ( Figure S2 ) or goat anti-rabbit immunoglobulin G (IgG)-Alexa568 secondary ( Figure S4 B). GS was detected using a mouse anti-GS monoclonal antibody (Millipore) and AF488-labeled secondary antibody. Slides were imaged on the Leica TCS SP8 confocal laser scanning microscope with a multiband spectral detector at 63× magnification using 488- and 552-nm lasers at 3.9% and 1.5% laser power, respectively. GS antibody signal (Alexa Fluor 488) was detected at 493- to 552-nm wavelengths, and GFP antibody signal (Alexa Fluor 568) was detected at 557- to 585-nm wavelengths using Leica hybrid photon detectors.

Cas9 Western Blot Mice were dosed with LNP containing Cas9 mRNA and single guide targeting TTR. After 7 days, mice were sacrificed, and liver biopsies were snap frozen. Liver tissue was lysed in RIPA buffer containing protease inhibitors. 30 μg protein was separated on 4%–12% Bis-tris Gel (Invitrogen) and transferred to nitrocellulose membranes in transfer buffer (Boston BioProducts) containing 20% methanol. Cas9 was detected with rabbit polyclonal anti-Cas9 (Santa Cruz), followed by goat anti-rabbit DyLight680 (Thermo Scientific), and detected on the Li-Cor Odyssey.

TTR IHC Cross sections of liver were collected from each mouse and fixed in 10% neutral buffered formalin for 24 hr. Tissue was transferred to 70% ethanol for shipping to Histotox Labs (Boulder, CO, USA). Livers were embedded, and blocks were sectioned to 3–5 μm and put on positively charged slides. Staining for TTR was performed using an automated Leica bond Rxm staining protocol. Paraffin embedded sections were dewaxed using a series of rinses with Bond Dewax solution (Leica, AR9222) for 30 s at 72°C followed by wash buffer rinses (Leica, AR9590). Bond Epitope Retrieval solution 1 (Leica, AR9961) was used for 20 min at 96°C followed by a series of wash buffer rinses. Dako Envision and a peroxide block were applied to the tissue for 5 min before rinsing. Protein block (Abcam, ab64226) was added for 10 min followed by αTTR (LSBio, LS-C407961) at a dilution of 1:250 for a 30-min incubation. After washing, the tissue was placed in Envision and Rabbit HRP Polymer (Dako, K4011) for 30 min followed by washing. Envision and DAB were added to the tissue for 5 min followed by hematoxylin for 5 min. Finally, the tissue was washed and placed on a covered slip. Images were scanned using the Aperio Digital Pathology software. Images were captured at a 10× magnification.