Genetic Editing Tools Research Articles

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

Illuminating the Cryptococcus neoformans species complex: unveiling intracellular structures with fluorescent-protein-based markers.

Cryptococcus neoformans is a fungal pathogen of the top critical priority recognized by the World Health Organization. This clinically important fungus also serves as a eukaryotic model organism. A variety of resources have been generated to facilitate investigation of the C. neoformans species complex, including congenic pairs, well-annotated genomes, genetic editing tools, and gene deletion sets. Here, we generated a set of strains with all major organelles fluorescently marked. We tested short organelle-specific targeting sequences and successfully labeled the following organelles by fusing the targeting sequences with a fluorescence protein: the plasma membrane, the nucleus, the peroxisome, and the mitochondrion. We used native cryptococcal Golgi and late endosomal proteins fused with a fluorescent protein to label these two organelles. These fluorescence markers were verified via colocalization using organelle-specific dyes. All the constructs for the fluorescent protein tags were integrated in an intergenic safe haven region. These organelle-marked strains were examined for growth and various phenotypes. We demonstrated that these tagged strains could be employed to track cryptococcal interaction with the host in phagocytosis assays. These strains also allowed us to discover remarkable differences in the dynamics of proteins targeted to different organelles during sexual reproduction. Additionally, we revealed that "dormant" spores transcribed and synthesized their own proteins and trafficked the proteins to the appropriate subcellular compartments, demonstrating that spores are metabolically active. We anticipate that these newly generated fluorescent markers will greatly facilitate further investigation of cryptococcal biology and pathogenesis.

HiPSC-derived cardiomyocytes as a model to study the role of small-conductance Ca2+-activated K+ (SK) ion channel variants associated with atrial fibrillation.

Atrial fibrillation (AF), the most common arrhythmia, has been associated with different electrophysiological, molecular, and structural alterations in atrial cardiomyocytes. Therefore, more studies are required to elucidate the genetic and molecular basis of AF. Various genome-wide association studies (GWAS) have strongly associated different single nucleotide polymorphisms (SNPs) with AF. One of these GWAS identified the rs13376333 risk SNP as the most significant one from the 1q21 chromosomal region. The rs13376333 risk SNP is intronic to the KCNN3 gene that encodes for small conductance calcium-activated potassium channels type 3 (SK3). However, the functional electrophysiological effects of this variant are not known. SK channels represent a unique family of K+ channels, primarily regulated by cytosolic Ca2+ concentration, and different studies support their critical role in the regulation of atrial excitability and consequently in the development of arrhythmias like AF. Since different studies have shown that both upregulation and downregulation of SK3 channels can lead to arrhythmias by different mechanisms, an important goal is to elucidate whether the rs13376333 risk SNP is a gain-of-function (GoF) or a loss-of-function (LoF) variant. A better understanding of the functional consequences associated with these SNPs could influence clinical practice guidelines by improving genotype-based risk stratification and personalized treatment. Although research using native human atrial cardiomyocytes and animal models has provided useful insights, each model has its limitations. Therefore, there is a critical need to develop a human-derived model that represents human physiology more accurately than existing animal models. In this context, research with human induced pluripotent stem cells (hiPSC) and subsequent generation of cardiomyocytes derived from hiPSC (hiPSC-CMs) has revealed the underlying causes of various cardiovascular diseases and identified treatment opportunities that were not possible using in vitro or in vivo studies with animal models. Thus, the ability to generate atrial cardiomyocytes and atrial tissue derived from hiPSCs from human/patients with specific genetic diseases, incorporating novel genetic editing tools to generate isogenic controls and organelle-specific reporters, and 3D bioprinting of atrial tissue could be essential to study AF pathophysiological mechanisms. In this review, we will first give an overview of SK-channel function, its role in atrial fibrillation and outline pathophysiological mechanisms of KCNN3 risk SNPs. We will then highlight the advantages of using the hiPSC-CM model to investigate SNPs associated with AF, while addressing limitations and best practices for rigorous hiPSC studies.

Efficient Biosynthesis of Acidic/Lactonic Sophorolipids and Their Application in the Remediation of Cyanobacterial Harmful Algal Blooms.

Cyanobacterial harmful algal blooms (CyanoHABs) pose significant threats to human health and natural ecosystems worldwide, primarily caused by water eutrophication, increased surface water temperature, and co-occurring microorganisms. Urgent action is needed to develop an eco-friendly solution to effectively curb the proliferation of CyanoHABs. Sophorolipids (SLs) are fully biodegradable biosurfactants synthesized by Starmerella bombicola. They can be classified into lactone and acid types. The lactone type displays strong antimicrobial activity, while the acid type exhibits good solubility, which make them ideal agents for mitigating CyanoHABs. Nevertheless, the broad utilization of SLs are hindered by their expensive production costs and the absence of effective genetic editing tools in the native host. In this study, we constructed recombinant strains capable of producing either acidic or lactonic SLs using the CRISPR-Cas9 gene editing system. The yields of acidic and lactonic SLs reached 53.64 g/L and 45.32 g/L in a shaking flask, respectively. In a 5 L fermenter, acidic SLs reached 129.7 g/L using low-cost glucose and rapeseed oil as substrates. The addition of 5 mg/L lactonic SLs effectively degraded cyanobacteria within 30 min, and a ratio of 8.25:1.75 of lactonic to acidic SLs showed the highest degradation efficiency. This study offers a safe and promising solution for CyanoHABs treatment.

Effect of Janus kinase (JAK) signaling inhibition on lineage plasticity and drug sensitivity in castrate resistant prostate cancer.

227 Background: Despite the remarkable successes of targeted cancer therapies, certain cancers, including lung, breast, and prostate cancer and melanoma, invariably become resistant to therapy. One mechanism of secondary resistance—lineage plasticity—arises when cells alter their identity and transition into aggressive states. In the case of prostate cancer, cells can acquire a neuroendocrine histology. This is often associated with a loss of tumor suppressor genes, such as TP53, RB1, and PTEN. However, while these genomic events initiate plasticity, tumor progression is not always associated with successive genomic alterations. This, in essence, not only poses a clinical challenge, but also confronts us with a wide-open biological question—what are the molecular underpinnings of lineage plasticity, and importantly, can the process be reversed? Methods: To study the temporal evolution of lineage plasticity and its relationship to androgen receptor signaling inhibitor (ARSI) resistance, we utilized genetically engineered mouse models (GEMMs) and murine organoids that were deleted for Tp53, Rb1, and/or Pten. Single cell RNA analyses were utilized to dissect which genes and pathways were up-regulated and most associated with the progression of plasticity. Plasticity-associated genes and pathways were perturbed using FDA-approved inhibitors or genetic editing tools. The presence of these pathways was confirmed in a subset of metastatic index lesions obtained by radiologically guided biopsies and as visualized by metabolic imaging. Furthermore, relevant findings were functionally validated in human tumor derived organoids (“tumoroids”). Results: Using GEMMs and organoid models, we found the lineage plasticity depended on increased Janus Kinase (JAK) and fibroblast growth factor receptor (FGFR) activity. Pharmacologic inhibition using FDA–approved inhibitors of JAK/STAT (ruxolitinib) and FGFR (erdafinitib), or through genetic knockdown, demonstrated increased androgen receptor (AR) signaling and restored ARSI sensitivity. These findings were further validated in a subset of ARSI-insensitive human tumoroids. By performing single cell RNA sequencing on mCRPC tumors biopsies, the presence of highly plastic JAK/STAT- and FGFR-high cells were confirmed, with implications for stratifying patients for clinical trials. Conclusions: JAK/STAT and FGFR signaling pathways promote lineage plasticity and result in complete insensitivity to androgen receptor signaling inhibitors (ARSIs). FDA-approved inhibitors of JAK/STAT (ruxolitinib) and FGFR (erdafitinib) synergize to reverse lineage plasticity and restore ARSI sensitivity.

Genomic Editing and Diabetes.

The diabetes types and its complications have varying developmental and metabolic pathways. There is an interplay of nongenetic and genetic components in pathogenesis of diabetes and its complications. There are several established genes such as ABCC8, TCF7L2, SLC2A2, and CAPN10 which are known to influence blood insulin and glucose levels. Current management of diabetes types may include lifetime burdensome use of insulin, insulin sensitizers, insulin secretagogues, etc. There has been increasing interest in improving genetic editing tools such as CRISPR/Cas9 and using genetically edited stem cells to alter diabetes disease course or possibly cure it. Current research on microRNAs and long noncoding RNAs may provide insights into the pathways involved in development of diabetes and its complications. Consequently, developing further understanding of genetics and its messenger pathways in diabetes would enhance our ability to develop precise and accurate genetic editing tools which can translate into clinically useful therapeutics.

Evaluation of Human Performance Aiding Live Synthetically Engineered Bacteria in a Gut-on-a-Chip.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E.coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 μM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.

New therapeutics for primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder that causes hepatic overproduction of oxalate and, often, nephrocalcinosis, nephrolithiasis, chronic kidney disease, and kidney failure. The purpose of the review is to provide an update on current emerging therapies for the treatment of PH1. Use of ribonucleic acid interference (RNAi) therapeutics that target the liver to block production of key enzymes along pathways that generate oxalate is a promising approach. Available evidence supports the efficacy of both Lumasiran (targeting glycolate oxidase) and Nedosiran (targeting hepatic lactate dehydrogenase (LDHa)) to reduce urinary oxalate excretion in PH1. The efficacy of alternative approaches including stiripentol (an anticonvulsant drug that also targets LDHa), lanthanum (a potential gastrointestinal oxalate binder), and Oxalobacter formigenes (a bacterium that can degrade oxalate within the gastrointestinal tract and may also increase its secretion from blood) are all also under study. Genetic editing tools including clustered regularly interspaced short palindromic repeats/Cas9 are also in preclinical study as a potential PH1 therapeutic. Novel treatments can reduce the plasma oxalate concentration and urinary oxalate excretion in PH1 patients. Thus, it is possible these approaches will reduce the need for combined kidney and liver transplantation to significantly decrease the morbidity and mortality of affected patients.

Modelling of primary ciliary dyskinesia using patient-derived airway organoids

Patient-derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long-term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient-specific differences in ciliary beating are observed and are in agreement with the patients' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient-specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.

Bioengineering methods for organoid systems.

Organoids have been widely used in fundamental, biomimetic, and therapeutic studies. These multicellular systems form via cell-autonomous self-organization where a cohort of stem cells undergoes in vivo-like proliferation, differentiation, and morphogenesis. They also recapitulate a series of physiological cell organization, complexity and functions that are untouchable by conventional bio-model systems using immortal cell lines. However, the development of organoids is often not easily controlled and their shape and size are yet fully physiological. Recent research has demonstrated that multiple bioengineering tools could be harnessed to control important internal and external cues that dictate stem cell behavior and stem-cell based organoid development. In this review, we introduce the current development of organoid systems and their potentials, as well as their limitations that impede their further utility in research and clinical fields. In comparison to conventional autonomous organoid system, we then review bioengineering approaches that offer improved control over organoid growth and development. We focus on the genetic editing tools that allow the program of build-in responses and phenotypes for organoid systems with enhanced physiological relevance. We also highlight the advances in bioengineering methods to modify cellular external milieus to generate desirable cell composition, 3D micro-architectures, and complex microfluidic systems. We conclude that the emerging biomimetic methods that employ multidisciplinary approaches could prevail in the future development of organoid systems.

Recombineering and MAGE.

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering.

Engineered Akkermansia muciniphila: A promising agent against diseases (Review).

Achieving a harmonious gut microbial ecosystem has been hypothesized to be a successful method for alleviating metabolic disorders. The administration of probiotics, such as Lactobacillus and Bifidobacteria, is a known traditional and safe pathway to regulate human commensal microbes. With advancements in genetic sequencing and genetic editing tools, more bacteria are able to function as engineered probiotics with multiple therapeutic properties. As one of the next-generation probiotic candidates, Akkermansia muciniphila (A. muciniphila) has been discovered to enhance the gut barrier function and moderate inflammatory responses, exhibit improved effects with pasteurization and display beneficial probiotic effects in individuals with obesity, type 2 diabetes, atherosclerosis and autism-related gastrointestinal disturbances. In view of this knowledge, the present review aimed to summarize the effects of A. muciniphila in the treatment of metabolic disorders and to discuss several mature recombination systems for the genetic modification of A. muciniphila. From gaining an enhanced understanding of its genetic background, ingested A. muciniphila is expected to be used in various applications, including as a diagnostic tool, and in the site-specific delivery of therapeutic drugs.

Methodology for Efficient and Biosecure Genetic Engineering of Biomedical Swine Models

The genetic proximity of swine to human, the overwhelming anatomical, physiological and pathophysiological similarities, and the ability to mimic external factors in a practical manner make swine the ideal preclinical model for many human diseases. While the value of swine in biomedical research is well recognized, their use has been limited by the lack of tools to easily edit the swine genome to model human disease, and the lack of facilities, resources and expertise at most institutions for supporting large‐scale swine research. The recent implementation of new genetic editing tools such as the CRISPR/Cas9 platform make the creation of novel genetic swine models possible and practical. However, conventional approaches to genetic engineering of swine rely on ova obtained from commercial abattoirs, significantly compromising biosecurity, and often offering little control over the background genetic of the swine breeds upon which the genomic editing is performed. Here, we describe methods and workflow that we have developed that allow for efficient genetic engineering of swine, while maintaining control over background genetics and minimizing the biosecurity risk to the animals and the facility. The process centrally relies on the refinement of reproductive cycle synchronization and superovulation of swine and time‐precise coordination of the entire genetic engineering workflow.Support or Funding InformationThe research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF North Central, NF Network, and NF Team Foundation

Abstract IA13: Reprogramming the epigenome for enhancing CAR T-cell antitumor efficacy

Abstract Many studies indicate that adoptive cell transfer therapy with autologous chimeric antigen receptor (CAR) T cells is a powerful approach for the treatment of cancer. CAR-modified T cells have shown activity in several clinical trials including our own at the University of Pennsylvania, with complete response rates as high as 80-90% in certain malignancies. However, the effectiveness of this approach for cancers involving T-cell defects, such as multiple myeloma, pancreatic cancer, and chronic lymphocytic leukemia (CLL), is much lower. The key questions currently facing the field are how to: i) enhance the potency and sustain the in vivo function of CAR T cells for such indications and ii) develop strategies to increase the resistance of CAR T cells to intrinsic and extrinsic dysfunction (e.g., immunosenescence, exhaustion, or other disease-specific defects). Because cell fate determination programs encoded in DNA are interpreted, modified, transmitted, and expanded as chromatin, we hypothesize that immunotherapy and reversal of T-cell dysfunction in cancer can be improved by broadly or specifically targeting appropriate epigenetic pathways. In this vein, we explore manipulation of the “writers” and “readers” of the histone code using genetic editing tools (e.g., CRISPR/Cas9) and chemical modifiers to convert poorly functional T cells to a state that renders these lymphocytes effective at tumor elimination. The basic understanding of T-cell biology in the context of epigenetic regulation may result in enhancement of cellular immunotherapy strategies, including the discovery of several actionable improvements to CAR T-cell therapy. Citation Format: Joseph A. Fraietta. Reprogramming the epigenome for enhancing CAR T-cell antitumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr IA13.

Gene Therapy: An Outstanding Technique For Diseases

Nearly three decades ago, the first clinical trial based on gene therapy revolutionized the scientific field by demonstrating that it was possible to genetically modify harmful or defective cells and therapeutically improve patient conditions. Since then, this technique has been implemented for the study and treatment of different diseases, many of them fatal, with the hope of achieving a "cure". Several genetic editing tools have been developed and continue to be studied to improve the effectiveness of gene therapy, in addition to a more exhaustive analysis for choosing the type of vector, which is the main cause of adverse effects. In this review, we present characteristics of the gene therapy mechanism along with the types of vectors that are used in this procedure, followed by the most important applications in the medical field and briefly we describe some limitations and prospects to study in the future.

Integrated analysis of the critical region 5p15.3-p15.2 associated with cri-du-chat syndrome.

Cri-du-chat syndrome (CdCs) is one of the most common contiguous gene syndromes, with an incidence of 1:15,000 to 1:50,000 live births. To better understand the etiology of CdCs at the molecular level, we investigated theprotein–protein interaction (PPI) network within the critical chromosomal region 5p15.3–p15.2 associated with CdCs using systemsbiology. Data were extracted from cytogenomic findings from patients with CdCs. Based on clinical findings, molecular characterization of chromosomal rearrangements, and systems biology data, we explored possible genotype–phenotype correlations involving biological processes connected with CdCs candidate genes. We identified biological processes involving genes previously found to be associated with CdCs, such as TERT, SLC6A3, and CTDNND2, as well as novel candidate proteins with potential contributions to CdCs phenotypes, including CCT5, TPPP, MED10, ADCY2, MTRR, CEP72, NDUFS6, and MRPL36. Although further functional analyses of these proteins are required, we identified candidate proteins for the development of new multi-target genetic editing tools to study CdCs. Further research may confirm those that are directly involved in the development of CdCs phenotypes and improve our understanding of CdCs-associated molecular mechanisms.

Edición genética y comunicación

Communication is essential in all areas of society, but science is one of the inescapable keys. Communicating is sharing, showing, teaching, transferring discoveries, observations, and findings both to colleagues and to society in general. That’s why good communication must always accompany good science. CRISPR genetic editing tools allow us to modify at will the genome of any living organism, including our species. In this text I review different relevant communicative events in the short but intense life of these molecular scissors, so called for their ability to cut the DNA molecule effectively and with precision.

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit

BackgroundPlasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation.ResultsTo bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I-SceI and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing.ConclusionsOur novel EXIT circuit, which exploits the homing endonuclease I-SceI, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing.

Treatment of genetic disorders-A vision coming into focus.

This issue of the Seminar Series is devoted to reviewing the state-of-the-art for treatment of non-metabolic genetic disorders. We begin with a primer on the design of studies in rare diseases. We then review a broad spectrum of disorders to reflect many different genetic mechanisms, including disorders stemming from a chromosomal basis as well as those due to single gene aberrations; those demonstrating principles of imprinting and mosaicism; and single organ as well as multisystem disorders. In doing so, a wide variety of treatment approaches are explained in this issue, including cell therapy, chromosome therapy, gene therapy, strategies for gene regulation with exon skipping and anti-sense oligonucleotides, stem cell modification and reintroduction, and use of genetic editing tools. The number of disorders amenable to treatment with existing methods is expanding rapidly, and is sure to increase as new strategies and tools emerge. The field of clinical genetics will be instrumental in ushering these new technologies into the realm of patient care. © 2016 Wiley Periodicals, Inc.

Mutation and Human Disease. Hunting Mutations, Targeting Disease. Introduction.

![Figure][1] Genomic data centers that help connect human mutations to illness continue to expand. This one at the Sanger Institute in Hinxton, U.K., currently has 16 petabytes of DNA sequence information on file. PHOTO: GENOME RESEARCH LIMITED As sequencing technology races toward ever cheaper, faster, and more accurate ways to read an individual's entire genome, understanding the health implications of variations in the genetic code becomes more crucial. This genetic variation can be as small as a single nucleotide base-pair change, insertion, or deletion, or as large as the gain or loss of multiple chromosomes. Genetic mutations can cause suffering and early death. It is thus vital to understand their origins and how they affect biological function. Some deleterious mutations may arise and be maintained over generations within populations. These may only come to light when an individual carries two copies, as for a recessive Mendelian trait. Others may be exposed as soon as they arise within an individual. Such mutations can cause developmental defects if they occur very early in life, or contribute to aging and disease, notably cancer, in later life. Recent technological advances have made it easier to detect human mutations and link them to pathology. Our ability to identify and target cancer-specific mutations has opened up a new and promising line of research. The rise of new genetic editing tools and a deeper understanding of the genetic architecture of traits, combined with reproductive technologies, offer the possibility to eliminate or mitigate the deleterious impact of mutations. While some mutations are easier to target than others, which can cause frustration at the pace of translating genetic information into medical help, enormous progress has arguably been made in the decade since the first human genome was decoded—and there's the promise of even more clinical applications in the future. [1]: pending:yes