Applications Of Crispr Technologies In Research And Beyond Pdf

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CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. The technique is considered highly significant in biotechnology and medicine as it allows for the genomes to be edited in vivo with extremely high precision, cheaply and with ease.

Everything You Need to Know About CRISPR-Cas9

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells.

Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research.

Recent progress in developing programmable nucleases, such as zinc-finger nucleases ZFNs , transcription activator-like effector nucleases TALENs and clustered regularly interspaced short palindromic repeat CRISPR —Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice.

Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology. Over the last few years, the exuberant development of genome editing has revolutionized research on the human genome, which has enabled investigators to better understand the contribution of a single-gene product to a disease in an organism.

In the s, the development of genetic engineering manipulation of DNA or RNA established a novel frontier in genome editing. Gene disruption by targeting the locus with NHEJ leads to the formation of indels. When two DSBs target both sides of a pathogenic amplification or insertion, a therapeutic deletion of the intervening sequences can be created, leading to NHEJ gene correction. Historically, homologous recombination HR , in which undamaged homologous DNA fragments are used as templates, has been the approach to realize targeted gene addition, replacement, or inactivation; however, the utility of HR is heavily limited due to its inefficiency in mammalian cells and model organisms.

This mechanism may be used to introduce precise mutations by delivering an appropriately designed repair template into targeted cells directly, 9 , 10 thereby, in a site-specific manner, resulting in mutation correction or new sequence insertion.

Alternatively, NHEJ-mediated repair tends to result in errors because it leads to efficient formation of gene insertion or deletion indels in diverse lengths at the DSB site, which eventually causes gene inactivation. In the early development stage of genome editing, to induce the desired DSBs at each particular DNA target site, the engineering of distinct zinc-finger nucleases ZFNs 14 or meganucleases 15 has been the research focus. These nuclease systems required specialized competence to generate artificial proteins consisting of customizable sequence-specific DNA-binding domains, each connected to a nonspecific nuclease for target cleavage, providing researchers with unprecedented tools to perform genetic manipulation.

However, the main challenges for transcription activator-like effector nucleases TALEN approaches are the design of a complex molecular cloning for each new DNA target and its low efficiency of genome screening in successfully targeted cells.

The advent of programmable nucleases has greatly accelerated the proceedings of gene editing from concept to clinical practice and unprecedentedly enabled scientists with a powerful tool to maneuver literally any gene in a wide variety of cell types and species. Current preclinical research on genome editing primarily concentrates on viral infections, cardiovascular diseases CVDs , metabolic disorders, primary defects of the immune system, hemophilia, muscular dystrophy, and development of T cell-based anticancer immunotherapies.

Finally, we outline the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology. ZFNs are assembled by fusing a non-sequence-specific cleavage domain to a site-specific DNA-binding domain that is loaded on the zinc finger.

The target sequence recognition and specificity of ZFNs are determined by three major factors: a the amino acid sequence of each finger, b the number of fingers, and c the interaction of the nuclease domain. By virtue of the modular structure of ZFNs, both the DNA-binding and catalytic domains can be individually optimized, which enables scientists to develop novel modular assembly with sufficient affinity and specificity for genome engineering.

This DNA-binding domain consists of a highly conserved repeat sequence from transcription activator-like effector TALE , which is a protein originally discovered in the phytopathogenic Xanthomonas bacteria that naturally alters the transcription of genes in host plant cells. The amino acid sequence of each repeat is structurally similar, except for two hypervariable amino acids the repeat variable di-residues or RVDs at positions 12 and Following pioneering works on zinc-finger proteins, multiple effector domains have become accessible to support the fusion of TALE repeats for different genomic modification purposes, including nucleases, 37 transcriptional activators, 18 and site-specific recombinases.

The editing of DNA means the irreversible permanent change of genome information, and this process is also facing inevitable security risks and ethical problems. As a result, genome editing strategies that only edit and modify RNA have also been proposed by scientists. With the use of CRISPR technology, RNA mutation is modified briefly, which not only avoids the irreversible modification of the genome but also can repair protein function in almost all cells to treat a variety of diseases.

It has been proven that patient-induced pluripotent stem cells iPSCs have the ability to differentiate into retinal precursors, and it is a useful cell source for cell replacement therapy without immune rejection problems. Targeted gene modification via chimeric genome editing tools e. These genome editing tools have enabled investigators to use genetically engineered animals to understand the etiology behind various diseases and to clarify molecular mechanisms that can be exploited for better therapeutic strategies Fig.

Ex vivo and in vivo genome editing for clinical therapy. Right: For in ex vivo editing therapy, cells are isolated from a patient to be treated, edited and then re-engrafted back to the patient. To achieve therapeutic success, the target cells must be able to survive in vitro and return to the target tissue after transplantation.

Left: For in vivo editing therapy, engineered nucleases are delivered by viral or nonviral approaches and directly injected into the patient for systemic or targeted tissue such as the eye, brain, or muscle effect.

Oncogenes and mutant tumor suppressor genes provide outstanding opportunities for the use of genome modulating approaches. As an archetypal platform for programmable DNA cleavage, ZFN-mediated targeting has been successfully applied to modify many genes in human cells and a number of model organisms, thus opening the door to the development and application of genome editing technologies.

ZFN-driven gene disruption was primarily demonstrated in when a three-finger protein was constructed to specifically block the expression of the BCR-ABL human oncogene that was transformed into a mouse cell line. A yeast-one-hybrid Y1H four-finger ZFN was designed to replace mutant p53 with wild-type p53 in several cancer cell lines from glioblastoma, leukemia and breast cancer via ZFN-induced HR. In addition to modifying viral genes associated with tumorigenesis, researchers have applied ZFNs to optimize T cell-mediated antitumor therapy.

To achieve this goal, Reik et al. This technology has recently been effective in knocking out glucose transport-related genes MCT4 or BSG in two glycolytic tumor models: colon adenocarcinoma and glioblastoma. Recent studies have also shown that TALEN gene editing technology used to knock out genes in cancer cells including cells from prostate cancer, 76 breast cancer, 77 and hepatocellular carcinoma HCC 78 is a powerful and broadly applicable platform to explore gene mutations at the molecular level.

Such an approach has already been applied in a study modeling tumor progression with an adenoma-carcinoma-metastasis sequence. CVD is a serious hazard to human health and is the number one cause of death in many industrialized countries.

Many different types of CVD are usually associated with a single genetic mutation or a combination of rare inherited heterozygous mutations. Currently, the establishment of in vivo CVD models with gene editing technology and the in-depth analysis of CVD pathogenic genes as well as their molecular mechanisms have made it possible to test the ability of gene therapy to control specific gene expression and improve gene functions. With the help of genome editing technologies, various research models of cardiovascular conditions have been created.

Abrahimi et al. It is promising to apply such technology in the field of allograft bioengineering, including the refinement of heart transplant. In this transgenic model, high levels of Cas9 were expressed exclusively in heart cardiomyocytes.

The investigators then intraperitoneally injected sgRNA targeting Myh6 loaded in an adeno-associated virus AAV vector, subsequently inducing cardiac-specific gene modification at the Myh6 locus, finally leading to hypertrophic cardiomyopathy. It has been demonstrated in the whole-exome sequencing of a nuclear family that three missense variants of a single nucleotide in the MKL2, MYH7, and NKX genes pass on to three offspring with cardiomyopathy with childhood onset.

An analysis of mouse heart and human induced pluripotent stem-cell-derived cardiomyocytes provides histological and molecular evidence for the contribution of the NKX variant as a genetic modifier. Porcine models resemble human conditions by physiology, anatomy, and genetics and are often considered ideal models for human cardiovascular structure research. Yang et al. Marfan syndrome MFS is an autosomal dominant disease caused by a mutation of heterozygous fibrillin-1 FBN1 and presents cardiovascular symptoms and skeletal abnormalities.

By the same principle, Umeyama et al. By genome editing techniques, potential therapeutic methods of repairing disease-causing mutations or of knocking out specific genes as CVD prevention approaches have also received widespread attention. Hybrid mutations in multiple genes may lead to LQTS, some of which have relatively clear mutation sites with known molecular functions, such as hERG gene mutations in the pore-forming subunit alpha protein that encodes the potassium voltage-gated channel.

The hERG gene mainly expresses and functions in cells of the myocardium and smooth muscle, and its mutation can cause fatal ventricular arrhythmia. Since these two genes are mainly expressed in liver cells, one idea is to directly introduce nonsense mutations to APOC3 or PCSK9 genes in liver cells through genome editing technology, thus fundamentally inhibiting protein synthesis and achieving long-term stable therapeutic effects.

Metabolic diseases include a group of syndromes that are caused by both genetic factors and the environment. Leptin Lep is a hormone secreted by white fat cells that acts on the metabolic regulation center of the hypothalamus through the leptin receptor LepR. Bao et al. Homozygous LepR-deficient mice are characterized by obesity, hyperphagia, hyperglycemia, insulin resistance, and lipid metabolism disorders, together with some complications of diabetes.

FTO gene mutations inhibit the conversion of white fat to brown fat. Progeny F1 mice were histologically labeled as Cre-loxP recombination, which was observed in all islets expressing insulin-positive cells and negatively expressed in other tissues. There was no significant difference in glucose tolerance between these genetically edited mice and wild-type mice.

Gene editing technology is also critically involved in the study of lipid metabolism. Nakagawa et al. The evidence above provides a new understanding of the role of CREB3L3 in plasma triglyceride metabolism and its contribution to liver and intestinal cholesterol metabolism.

In , Carlson et al. Recent genome-wide association studies have identified tribble homolog 1 TRIB1 to be associated with lipoprotein metabolism in human hepatocytes. These mechanisms indicate that NDs are induced by complicated interactions of multiple genetic factors; either alone or in combination, the interactions lead to clinical features.

The emergence of gene editing platforms provides a convenient approach to study gene functions related to NDs. As a promising breakthrough in the field of NDs, the development of HTT gene knock-in pigs would be of great significance for pathogenesis research and therapy exploration in Huntington disease.

Mutations in the gene encoding amyloid precursor protein APP cause familial AD with nearly complete penetrance. These transgenic cells can be used to elucidate aspects of the molecular mechanisms of AD pathogenesis, particularly those involved in the mutant amyloidogenic pathway affecting the APP coding sequence. After correction, the mtDNA damage disappeared in differentiated neural progenitor and neural cells derived from iPSCs.

This system has identified PD-associated risk variants in noncoding distal enhancer elements that regulate SNCA expression; it has also confirmed that the transcriptional disorder of SNCA is related to sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX These results suggest that gene editing techniques can generate specific ND animal models for further exploration into human diseases, and they are potentially capable of offering a robust therapeutic approach against multiple human genetic defects that have been considered incurable.

Gene editing platforms have emerged recently as antiviral therapeutics for treating infectious diseases, either by altering the host genes required by the virus or by targeting the viral genes necessary for replication. In , a patient was functionally cured of HIV infection by transplanting allogeneic stem cells from a donor with a homozygous CCR5 d32 allele, suggesting that it is feasible to obtain resistance to HIV by mimicking natural homozygous CCR5 d32 mutations with genome editing technologies.

Laboratory results from Ebina and Hu et al. Similarly, Hendel et al. The sustained expression of high-risk human papillomavirus HPV oncogenes E6 and E7 is implicated in malignant transformation and is strongly associated with cervical cancer. Ding et al. When different plasmid-encoded zinc-finger modules were introduced in vivo, the therapeutic effects of ZFNs were further confirmed, inhibiting tumor growth in mice bearing cervical cancer cells. Similar results in another study showed that using ZFNs to target HPV E7 induced specific shear of the E7 gene and attenuated its malignant biological effect.

Hepatitis B virus HBV is the most important pathogen of liver disease. Genomic editing technology allows us to gain a deeper understanding of the mechanisms underlying variant diseases associated with viral infection and demonstrates tremendous potential in the development of therapeutic approaches against viral infections, which represent some of the most intractable diseases. In recent years, with the advancement of gene sequencing technology, it is more explicit to make the genetic diagnosis of a variety of hereditary eye diseases, such as congenital cataract, congenital glaucoma, retinitis pigmentosa RP , congenital corneal dystrophy, Leber congenital amaurosis LCA , retinoblastoma RB , and Usher syndrome.

Moreover, they created a knock-in mouse model of Reep6 p. The rodless rd1 mouse, the most vastly used preclinical model of RP, has been aggressively debated for nearly a century after its occurrence because the cause of the blinding RP phenotype remains undetermined. The rd1 mouse has two homozygous variants in the Pde6b locus of chromosome 5: a nonsense mutation YX and a murine leukemia virus Xmv insertion in the reverse orientation in intron 1.

Benjamin et al.

Application and Development of CRISPR/Cas9 Technology in Pig Research

What is it? And why is the scientific community so fascinated by its potential applications? Starting with its definition, we explain how this technology harnesses an ancient bacteria-based defense system — and how it will impact the world around us today. Imagine a future where parents can create bespoke babies, selecting the height and eye color of their unborn children. Download the entire page free report PDF to learn how and when.

CRISPR gene editing

The applications of this technology are limitless. Scientists are also using CRISPR to develop more sustainable methods for producing fuel and manufacturing chemicals, and to improve food crops so we can better feed the growing global population. Now that CRISPR has gained popularity within the scientific community, there is a surplus of information surrounding this technology. The snipped DNA fragment may then be stored between the palindromic CRISPR sequences to retain a genetic memory for disabling future infections from the same viral strain. Once scientists learned how the CRISPR system worked in bacteria, they figured out how to reprogram it to allow efficient editing in any species.

Genomic editing to correct disease-causing mutations is a promising approach for the treatment of human diseases. Here, we review the application of the CRISPR system over the last 2 years; including its development and application in base editing, transcription modulation and epigenetic editing, genomic-scale screening, and cell and embryo therapy. In recent years, this system has garnered increasing attention as an effective and simple genome-engineering tool and has revolutionized the life sciences.

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Barrangou and Doudna, ; Wang and Qi, Because its targeting precision and efficiency have been well studied, targeting sites can be designed to minimize off-target binding and maximize on-target efficiency Hsu et al. In addition, based on the structure of Cas9, rationally engineered SpCas9-NG variant, also with an increased targeting range higher activity towards NGD sites than NGC sites , promises a better efficiency over xCas9 at NGD targeting sites and suggests a way to design Cas9 for further optimization Nishimasu et al. Utilizing the concept, we could repair the mutations that induce diesease, which propose a bright future in gene therapy Wu et al.

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