Mpox infection protects against re-challenge in rhesus macaques.

The mpox outbreak of 2022-2023 involved rapid global spread in men who have sex with men. We infected 18 rhesus macaques with mpox by the intravenous, intradermal, and intrarectal routes and observed robust antibody and T cell responses following all three routes of infection. Numerous skin lesions and high plasma viral loads were observed following intravenous and intradermal infection. Skin lesions peaked on day 10 and resolved by day 28 following infection. On day 28, we re-challenged all convalescent and 3 naive animals with mpox. All convalescent animals were protected against re-challenge. Transcriptomic studies showed upregulation of innate and inflammatory responses and downregulation of collagen formation and extracellular matrix organization following challenge, as well as rapid activation of T cell and plasma cell responses following re-challenge. These data suggest key mechanistic insights into mpox pathogenesis and immunity. This macaque model should prove useful for evaluating mpox vaccines and therapeutics.

Vaccine protection against the SARS-CoV-2 Omicron variant in macaques.

The rapid spread of the SARS-CoV-2 Omicron (B.1.1.529) variant, including in highly vaccinated populations, has raised important questions about the efficacy of current vaccines. In this study, we show that the mRNA-based BNT162b2 vaccine and the adenovirus-vector-based Ad26.COV2.S vaccine provide robust protection against high-dose challenge with the SARS-CoV-2 Omicron variant in cynomolgus macaques. We vaccinated 30 macaques with homologous and heterologous prime-boost regimens with BNT162b2 and Ad26.COV2.S. Following Omicron challenge, vaccinated macaques demonstrated rapid control of virus in bronchoalveolar lavage, and most vaccinated animals also controlled virus in nasal swabs. However, 4 vaccinated animals that had moderate Omicron-neutralizing antibody titers and undetectable Omicron CD8+ T cell responses failed to control virus in the upper respiratory tract. Moreover, virologic control correlated with both antibody and T cell responses. These data suggest that both humoral and cellular immune responses contribute to vaccine protection against a highly mutated SARS-CoV-2 variant.

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Low-dose Ad26.COV2.S protection against SARS-CoV-2 challenge in rhesus macaques.

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26)-vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. To evaluate reduced doses of Ad26.COV2.S, 30 rhesus macaques were immunized once with 1 × 1011, 5 × 1010, 1.125 × 1010, or 2 × 109 viral particles (vp) Ad26.COV2.S or sham and were challenged with SARS-CoV-2. Vaccine doses as low as 2 × 109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125 × 1010 vp were required for protection in nasal swabs. Activated memory B cells and binding or neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show enhancement of disease. These data demonstrate that a single immunization with relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques, although a higher vaccine dose may be required for protection in the upper respiratory tract.

mRNA-1273 protects against SARS-CoV-2 beta infection in nonhuman primates.

B.1.351 is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant most resistant to antibody neutralization. We demonstrate how the dose and number of immunizations influence protection. Nonhuman primates received two doses of 30 or 100 µg of Moderna's mRNA-1273 vaccine, a single immunization of 30 µg, or no vaccine. Two doses of 100 µg of mRNA-1273 induced 50% inhibitory reciprocal serum dilution neutralizing antibody titers against live SARS-CoV-2 p.Asp614Gly and B.1.351 of 3,300 and 240, respectively. Higher neutralizing responses against B.1.617.2 were also observed after two doses compared to a single dose. After challenge with B.1.351, there was ~4- to 5-log10 reduction of viral subgenomic RNA and low to undetectable replication in bronchoalveolar lavages in the two-dose vaccine groups, with a 1-log10 reduction in nasal swabs in the 100-µg group. These data establish that a two-dose regimen of mRNA-1273 will be critical for providing upper and lower airway protection against major variants of concern.

Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine.

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.

Correlates of protection against SARS-CoV-2 in rhesus macaques.

Recent studies have reported the protective efficacy of both natural1 and vaccine-induced2-7 immunity against challenge with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in rhesus macaques. However, the importance of humoral and cellular immunity for protection against infection with SARS-CoV-2 remains to be determined. Here we show that the adoptive transfer of purified IgG from convalescent rhesus macaques (Macaca mulatta) protects naive recipient macaques against challenge with SARS-CoV-2 in a dose-dependent fashion. Depletion of CD8+ T cells in convalescent macaques partially abrogated the protective efficacy of natural immunity against rechallenge with SARS-CoV-2, which suggests a role for cellular immunity in the context of waning or subprotective antibody titres. These data demonstrate that relatively low antibody titres are sufficient for protection against SARS-CoV-2 in rhesus macaques, and that cellular immune responses may contribute to protection if antibody responses are suboptimal. We also show that higher antibody titres are required for treatment of SARS-CoV-2 infection in macaques. These findings have implications for the development of SARS-CoV-2 vaccines and immune-based therapeutic agents.

Protective efficacy of Ad26.COV2.S against SARS-CoV-2 B.1.351 in macaques.

The emergence of SARS-CoV-2 variants that partially evade neutralizing antibodies poses a threat to the efficacy of current COVID-19 vaccines1,2. The Ad26.COV2.S vaccine expresses a stabilized spike protein from the WA1/2020 strain of SARS-CoV-2, and has recently demonstrated protective efficacy against symptomatic COVID-19 in humans in several geographical regions-including in South Africa, where 95% of sequenced viruses in cases of COVID-19 were the B.1.351 variant3. Here we show that Ad26.COV2.S elicits humoral and cellular immune responses that cross-react with the B.1.351 variant and protects against B.1.351 challenge in rhesus macaques. Ad26.COV2.S induced lower binding and neutralizing antibodies against B.1.351 as compared to WA1/2020, but elicited comparable CD8 and CD4 T cell responses against the WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C variants. B.1.351 infection of control rhesus macaques resulted in higher levels of virus replication in bronchoalveolar lavage and nasal swabs than did WA1/2020 infection. Ad26.COV2.S provided robust protection against both WA1/2020 and B.1.351, although we observed higher levels of virus in vaccinated macaques after B.1.351 challenge. These data demonstrate that Ad26.COV2.S provided robust protection against B.1.351 challenge in rhesus macaques. Our findings have important implications for vaccine control of SARS-CoV-2 variants of concern.

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma.

INTRODUCTION: Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterized by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. We aimed to identify genetic and morphological variation that can be attributed to trabecular meshwork cell (TMC) dysfunction and raised IOP in POAG. METHODS: 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescence coupled with CellProfiler image analysis allowed for single-cell morphological profiling. RESULTS: Many gene knockouts invoked DEGs relating to matrix metalloproteinases and interferon-induced proteins. We have prioritized genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified, suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, while knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSION: High-throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

Defining the determinants of protection against SARS-CoV-2 infection and viral control in a dose-down Ad26.CoV2.S vaccine study in nonhuman primates.

Despite the rapid creation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines, the precise correlates of immunity against severe Coronavirus Disease 2019 (COVID-19) are still unknown. Neutralizing antibodies represent a robust surrogate of protection in early Phase III studies, but vaccines provide protection prior to the evolution of neutralization, vaccines provide protection against variants that evade neutralization, and vaccines continue to provide protection against disease severity in the setting of waning neutralizing titers. Thus, in this study, using an Ad26.CoV2.S dose-down approach in nonhuman primates (NHPs), the role of neutralization, Fc effector function, and T-cell immunity were collectively probed against infection as well as against viral control. While dosing-down minimally impacted neutralizing and binding antibody titers, Fc receptor binding and functional antibody levels were induced in a highly dose-dependent manner. Neutralizing antibody and Fc receptor binding titers, but minimally T cells, were linked to the prevention of transmission. Conversely, Fc receptor binding/function and T cells were linked to antiviral control, with a minimal role for neutralization. These data point to dichotomous roles of neutralization and T-cell function in protection against transmission and disease severity and a continuous role for Fc effector function as a correlate of immunity key to halting and controlling SARS-CoV-2 and emerging variants.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.

Experimental laboratory models as tools for understanding modifiable dementia risk.

Experimental laboratory research has an important role to play in dementia prevention. Mechanisms underlying modifiable risk factors for dementia are promising targets for dementia prevention but are difficult to investigate in human populations due to technological constraints and confounds. Therefore, controlled laboratory experiments in models such as transgenic rodents, invertebrates and in vitro cultured cells are increasingly used to investigate dementia risk factors and test strategies which target them to prevent dementia. This review provides an overview of experimental research into 15 established and putative modifiable dementia risk factors: less early-life education, hearing loss, depression, social isolation, life stress, hypertension, obesity, diabetes, physical inactivity, heavy alcohol use, smoking, air pollution, anesthetic exposure, traumatic brain injury, and disordered sleep. It explores how experimental models have been, and can be, used to address questions about modifiable dementia risk and prevention that cannot readily be addressed in human studies. HIGHLIGHTS: Modifiable dementia risk factors are promising targets for dementia prevention. Interrogation of mechanisms underlying dementia risk is difficult in human populations. Studies using diverse experimental models are revealing modifiable dementia risk mechanisms. We review experimental research into 15 modifiable dementia risk factors. Laboratory science can contribute uniquely to dementia prevention.

A novel functional role for the classic CNS neurotransmitters, GABA, glycine, and glutamate, in the kidney: potent and opposing regulators of the renal vasculature.

The presence of a renal GABA/glutamate system has previously been described; however, its functional significance in the kidney remains undefined. We hypothesized, given its extensive presence in the kidney, that activation of this GABA/glutamate system would elicit a vasoactive response from the renal microvessels. The functional data here demonstrate, for the first time, that activation of endogenous GABA and glutamate receptors in the kidney significantly alters microvessel diameter with important implications for influencing renal blood flow. Renal blood flow is regulated in both the renal cortical and medullary microcirculatory beds via diverse signaling pathways. GABA- and glutamate-mediated effects on renal capillaries are strikingly similar to those central to the regulation of central nervous system capillaries, that is, exposing renal tissue to physiological concentrations of GABA, glutamate, and glycine led to alterations in the way that contractile cells, pericytes, and smooth muscle cells, regulate microvessel diameter in the kidney. Since dysregulated renal blood flow is linked to chronic renal disease, alterations in the renal GABA/glutamate system, possibly through prescription drugs, could significantly impact long-term kidney function.NEW & NOTEWORTHY Functional data here offer novel insight into the vasoactive activity of the renal GABA/glutamate system. These data show that activation of endogenous GABA and glutamate receptors in the kidney significantly alters microvessel diameter. Furthermore, the results show that these antiepileptic drugs are as potentially challenging to the kidney as nonsteroidal anti-inflammatory drugs.

Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates.

BACKGROUND: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates. METHODS: Nonhuman primates received 10 or 100 µg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens. RESULTS: The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID50) geometric mean titers of 501 in the 10-µg dose group and 3481 in the 100-µg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-µg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group. CONCLUSIONS: Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.).

Protective Efficacy of Gastrointestinal SARS-CoV-2 Delivery against Intranasal and Intratracheal SARS-CoV-2 Challenge in Rhesus Macaques.

Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Postpyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage fluid. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage fluid following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that postpyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques. IMPORTANCE SARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution, and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however, no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here, we report that oral administration of live SARS-CoV-2 in nonhuman primates may offer prophylactic benefits, but the formulation and route of administration will require further optimization.

Seeing Neurodegeneration in a New Light Using Genetically Encoded Fluorescent Biosensors and iPSCs.

Neurodegenerative diseases present a progressive loss of neuronal structure and function, leading to cell death and irrecoverable brain atrophy. Most have disease-modifying therapies, in part because the mechanisms of neurodegeneration are yet to be defined, preventing the development of targeted therapies. To overcome this, there is a need for tools that enable a quantitative assessment of how cellular mechanisms and diverse environmental conditions contribute to disease. One such tool is genetically encodable fluorescent biosensors (GEFBs), engineered constructs encoding proteins with novel functions capable of sensing spatiotemporal changes in specific pathways, enzyme functions, or metabolite levels. GEFB technology therefore presents a plethora of unique sensing capabilities that, when coupled with induced pluripotent stem cells (iPSCs), present a powerful tool for exploring disease mechanisms and identifying novel therapeutics. In this review, we discuss different GEFBs relevant to neurodegenerative disease and how they can be used with iPSCs to illuminate unresolved questions about causes and risks for neurodegenerative disease.

Approaches for the sensitive detection of rare base and prime editing events.

Precision chemistry entailing user-directed nucleotide substitutions and template-specified repair can be facilitated by base editing and prime editing, respectively. Recently, the diversification of adenine, cytosine, and prime editor variants obliges a considered, high-throughput evaluation of these tools for optimized, end-point applications. Herein, we outline novel, cost-effective and scalable approaches for the rapid detection of base editing and prime editing outcomes using gel electrophoresis. For base editing, we exploit primer mismatch amplification (SNP genotyping) for the gel-based detection of base editing efficiencies as low as 0.1%. For prime editing, we describe a one-pot reaction combining polymerase chain reaction (PCR) amplification of the target region with restriction digestion (restriction fragment length polymorphism; RFLP). RFLP enables the rapid detection of insertion or deletion events in under 2.5 h from genomic DNA extraction. We show that our method of SNP genotyping is amenable to both endogenous target loci as well as transfected, episomal plasmid targets in BHK-21 cells. Next, we validate the incidence of base and prime editing by describing Sanger sequencing and next-generation sequencing (NGS) workflows for the accurate validation and quantification of on-target editing efficiencies. Our workflow details three different methods for the detection of rare base and prime editing events, enabling a tiered approach from low to high resolution that makes use of gel electrophoresis, Sanger sequencing, and NGS.

Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye. Here we describe our optimized protocol for using CRISPR/Cas ribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs and homology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery via nucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifying iPSC clones for further characterization.