Gaining Insight into Mitochondrial Genetic Variation and Downstream Pathophysiology: What Can i(PSCs) Do?

Mitochondria are specialized organelles involved in energy production that have retained their own genome throughout evolutionary history. The mitochondrial genome (mtDNA) is maternally inherited and requires coordinated regulation with nuclear genes to produce functional enzyme complexes that drive energy production. Each mitochondrion contains 5-10 copies of mtDNA and consequently, each cell has several hundreds to thousands of mtDNAs. Due to the presence of multiple copies of mtDNA in a mitochondrion, mtDNAs with different variants may co-exist, a condition called heteroplasmy. Heteroplasmic variants can be clonally expanded, even in post-mitotic cells, as replication of mtDNA is not tied to the cell-division cycle. Heteroplasmic variants can also segregate during germ cell formation, underlying the inheritance of some mitochondrial mutations. Moreover, the uneven segregation of heteroplasmic variants is thought to underlie the heterogeneity of mitochondrial variation across adult tissues and resultant differences in the clinical presentation of mitochondrial disease. Until recently, however, the mechanisms mediating the relation between mitochondrial genetic variation and disease remained a mystery, largely due to difficulties in modeling human mitochondrial genetic variation and diseases. The advent of induced pluripotent stem cells (iPSCs) and targeted gene editing of the nuclear, and more recently mitochondrial, genomes now provides the ability to dissect how genetic variation in mitochondrial genes alter cellular function across a variety of human tissue types. This review will examine the origins of mitochondrial heteroplasmic variation and propagation, and the tools used to model mitochondrial genetic diseases. Additionally, we discuss how iPSC technologies represent an opportunity to advance our understanding of human mitochondrial genetics in disease.

Toward the Treatment of Inherited Diseases of the Retina Using CRISPR-Based Gene Editing

Inherited retinal dystrophies [IRDs] are a common cause of severe vision loss resulting from pathogenic genetic variants. The eye is an attractive target organ for testing clinical translational approaches in inherited diseases. This has been demonstrated by the approval of the first gene supplementation therapy to treat an autosomal recessive IRD, RPE65-linked Leber congenital amaurosis (type 2), 4 years ago. However, not all diseases are amenable for treatment using gene supplementation therapy, highlighting the need for alternative strategies to overcome the limitations of this supplementation therapeutic modality. Gene editing has become of increasing interest with the discovery of the CRISPR-Cas9 platform. CRISPR-Cas9 offers several advantages over previous gene editing technologies as it facilitates targeted gene editing in an efficient, specific, and modifiable manner. Progress with CRISPR-Cas9 research now means that gene editing is a feasible strategy for the treatment of IRDs. This review will focus on the background of CRISPR-Cas9 and will stress the differences between gene editing using CRISPR-Cas9 and traditional gene supplementation therapy. Additionally, we will review research that has led to the first CRISPR-Cas9 trial for the treatment of CEP290-linked Leber congenital amaurosis (type 10), as well as outline future directions for CRISPR-Cas9 technology in the treatment of IRDs.

Prediction of off-target effects in crispr/cas9 system by ensemble learning

Background: CRISPR/Cas9, a new generation of targeted gene editing technology with low cost and simple operation has been widely employed in the field of gene editing. The erroneous cutting of off-target sites in CRISPR/Cas9 is called off-target effect, which is also the biggest complication that CRISPR/Cas9 confronts in practical application. To be specific, the off-target effects could lead to unexpected gene editing results. Therefore, accurately predicting CRISPR/Cas9 off-target effect is a very important task. Predicting off-target effects of CRISPR/Cas9 by machine learning method is feasible, but most existing off-target tools did not pay close attention to the effects of gene encoding on prediction. Methods: We compared three encoding methods based on One-Hot and combined the gene sequence with four CRISPR/Cas9 off-target prediction tools to build an ensemble model with XGBoost, designated as XGBCRISPR. The grid search is employed to find the optimal parameters to achieve the best performance. Results: The performance is compared with existing tools based on the ROC value and PRC value. The experimental results show that the XGBCRISPR model is superior to the existing tools. Conclusion: The new model could achieve better prediction result than existing tools, but the accuracy of model can be improved further as many off-target scores appear.

Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Next-generation sequencing is now utilized to identify genetic abnormalities and develop gene therapy

The genomic size, complexity, heritability, and diversity of human primary genetic compartments vary. Although the nuclear genome's huge size ensures that hundreds of reported monogenic diseases appear in a range of conditions, germline abnormalities in the mitochondrial and nuclear genomes often generate developmental issues. Accumulation of somatic mutations in the nuclear genome causes cancer, and somatic mutations in mitochondria may contribute to aging. More broadly, the microbial metagenome develops largely after birth, and is marked throughout their lifetimes by much more diversity and diversity among individuals. Mitochondrial sequencing, clinical exome and full-genome sequencing, and 16S and unbiased microbiological sequencing have all become more widely available because of developments in DNA sequencing next-generation.These technologies discover genetic defects that can be addressed with gene therapy. Modern aided techniques of reproduction, such as mitochondrial replacement therapy and preimplantation diagnosis, may address complete genomic compartments in bulk, such as mitochondrial and nuclear genomes. Additive somatic cell gene therapies started with the invention of viral vectors to infect human somatic cells that could be cultured ex vivo, such as T cells, and rapidly advanced to in vivo applications employing viral pseudotypes with specific tissue tropisms. CRISPR/Cas9 and other targeted gene editing approaches that fix the specific causative mutation or gene at its endogenous locus have recently expanded the possibility for more refined ex vivo and in vivo gene therapies.DNA sequencing costs have decreased during the past two decades, hurrying to identify genetic diseases. Targeted gene editing progress has now enabled the synthesis and testing of specific therapeutic reagents to address direct and accessible genetic abnormalities, repeating these diagnostic accomplishments. Generalized methods for delivering customizable gene editing reagents to the cell type and genomic compartment of interest in the specific genetic disease of a patient are one of the major outstanding challenges to wide-spread gene therapy. Aside from direct genetic disease repair, recent methods for rapidly identifying synthetic genetic circuits capable of improving cellular function in diseases such as cancer and autoimmune hold the promise of future gene therapy in modified somatic cells.Genetic diseases are becoming more readily diagnosed in all human genetic compartments, and the next generation of gene therapy platforms targeting each compartment are preparing to give flexible, tailored curative medicines. The Mitochondrial genome, nuclear genome, and microbial metagenome are the three genetic compartments present in humans. Gene therapies for each of these compartments come into three categories: whole genome replacement or selection, non-focused insertion of new genetic information to compensate for genetic errors, and direct gene editing to correct causative genetic disorders. The mitochondrial and nuclear genomes are determined at conception, save for somatic mutations and the adaptive immune receptor repertoire, and remain stable throughout life.

CRISPR/Cas9-induced β-carotene Hydroxylase Mutation in Dunaliella Salina CCAP19/18

Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of CRISPR/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

Targeted Gene Editing in Porcine Spermatogonia

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.

Concepts of Molecular Plant Breeding and Genome Editing

Traditional plant breeding depends on spontaneous and induced mutations available in the crop plants. Such mutations are rare and occur randomly. By contrast, molecular breeding and genome editing are advanced breeding techniques that can enhance the selection process and produce precisely targeted modifications in any crop. Identification of molecular markers, based on SSRs and SNPs, and the availability of high-throughput (HTP) genotyping platforms have accelerated the process of generating dense genetic linkage maps and thereby enhanced application of marker-assisted breeding for crop improvement. Advanced molecular biology techniques that facilitate precise, efficient, and targeted modifications at genomic loci are termed as “genome editing.” The genome editing tools include “zinc-finger nucleases (ZNFs),” “transcription activator-like effector nucleases (TALENs),” oligonucleotide-directed mutagenesis (ODM), and “clustered regularly interspersed short palindromic repeats (CRISPER/Cas) system,” which can be used for targeted gene editing. Concepts of molecular plant breeding and genome editing systems are presented in this chapter.