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CRISPR/Cas9 and Cytidine Base-Editing Systems for Efficient Genome Engineering in Oleaginous Rhodococcus.
Oleaginous Rhodococcus strains can degrade diverse compounds and synthesize and accumulate large amounts of intracellular lipids, making them attractive platforms for biotechnological applications. However, efficient genome editing in Rhodococcus remains challenging, and new molecular tools are needed to advance the understanding of its metabolism, stress responses, and cellular physiology. Here, we describe the nonhomologous end joining (NHEJ) system in rhodococci and implement an efficient genome-editing system based on a CRISPR/Cas9 nuclease approach that utilizes this repair mechanism, eliminating the need for donor DNA templates. In addition, we report the first implementation of a cytidine base-editing (CBE) system, enabling precise single-nucleotide substitutions (C•G → T•A) in oleaginous Rhodococcus strains. Both strategies rely on a dual-plasmid CRISPR platform, resulting in two plasmid sets: pTipCas9/pCA71sgRNA and pTipBE/pCA71sgRNA. These systems enabled high rates of INDEL formation and C•G → T•A base conversions, with efficiencies of 70-80% and 75-85% at native genomic targets, respectively. Finally, for the CBE system, we implemented a Csy4-mediated sgRNA-processing module to support multiplex genome editing, enabling the simultaneous modification of multiple loci. Together, these tools outperform recombination-based approaches and will facilitate the study of complex metabolic pathways and the development of genetic strategies for biotechnological applications in Rhodococcus, while also being transferable to other actinobacteria.
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Efficient generation of homozygous substitutions in rice in one generation utilizing an rABE8e base editor.
A new deaminase, TadA8e, was recently evolved in the laboratory. TadA8e catalyzes DNA deamination over 1,000 times faster than ABE7.10. We developed a high-efficiency adenine base editor, rABE8e (rice ABE8e), combining monomeric TadA8e, bis-bpNLS and codon optimization. rABE8e had substantially increased editing efficiencies at NG-protospacer adjacent motif (PAM) and NGG-PAM target sequences compared with ABEmax. For most targets, rABE8e exhibited nearly 100% editing efficiency and high homozygous substitution rates in the specific editing window, especially at Positions A5 and A6. The ability to rapidly generate plant materials with homozygous base substitutions will benefit gene function research and precision molecular breeding.
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Strategic transgene-free approaches of CRISPR-based genome editing in plants.
Genome editing through the alteration of nucleotide sequence has already revolutionized the field of site-directed mutagenesis for a decade. However, research in terms of precision and efficacy in targeting the loci and reduction in off-target mutation has always been a priority when DNA is involved. Therefore, recent research interest lies in utilizing the same precision technology but results in non-transgenic. In this review article, different technological advancements have been explained, which may provide a holistic concept of and need for transgene-free genome editing. The advantage and lacunas of each technology have been critically discussed to deliver a transparent view to the readers. A systematic analysis and evaluation of published research articles implied that researchers across the globe are putting continuous efforts in this direction to eliminate the hindrance of transgenic regulation. Nevertheless, this approach has severe implications legitimate for mitigating the conflict of acceptance, reliability, and generosity of gene-editing technology and sustainably retorting to the expanding global population feeding challenges.
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CRISPR-Mediated Base Editing: Promises and Challenges for a Viable Oncotherapy Strategy.
Base editing technology, developed from the CRISPR/Cas9 system, is able to efficiently implement single-base substitutions at specific DNA or RNA sites without generating double-strand breaks with precision and efficiency. Point mutations account for 58% of disease-causing genetic mutations in humans, and single nucleotide variants are an important cause of tumorigenesis, and the advent of base editors offers new hope for the study or treatment of such diseases. Although it has some limitations, base editors have been continuously improved in terms of editing efficiency, specificity, and product purity since their development. In this review, we examine the main base editing technologies and discuss their applications and prospects in tumor research and therapy, as well as elaborate on their mode of delivery.
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CRISPR-Mediated Base Editing without DNA Double-Strand Breaks.
Targeting point mutations using CRISPR/Cas9 so far has required efficient homologous recombination (HR) and donor oligonucleotides. In a recent Nature paper, Komor and colleagues (2016) describe a way to make specific base changes that does not depend on HR or donor DNA and does not involve making double-strand breaks.
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Therapeutic application of the CRISPR system: current issues and new prospects.
Since its discovery, the Clustered Regularly Interspaced Short Palindromic Repeat (the CRISPR) system has been increasingly applied to therapeutic genome editing. Employment of several viral and non-viral vectors has enabled efficient delivery of the CRISPR system to target cells or tissues. In addition, the CRISPR system is able to modulate the target gene's expression in various ways, such as mutagenesis, gene integration, epigenome regulation, chromosomal rearrangement, base editing and mRNA editing. However, there are still limitations hindering an ideal application of the system: inefficient delivery, dysregulation of the delivered gene, the immune response against the CRISPR system, the off-target effects or the unintended on-target mutations. In addition, there are recent discoveries that have not been yet applied to CRISPR-mediated therapeutic genome editing. Here, we review the overall principles related to the therapeutic application of the CRISPR system, along with new strategies for the further application and prospects to overcome the limitations.
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What a Clinician Needs to Know About Genome Editing: Status and Opportunities for Inborn Errors of Immunity.
During the past 20 years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector-mediated gene addition, inborn errors of immunity are excellent candidate diseases for a corrective autologous hematopoietic stem cell gene editing strategy. Research on gene editing for inborn errors of immunity is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of inborn errors of immunity. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.
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Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases.
Collectively, genetic disorders affect approximately 350 million individuals worldwide and are a major global health burden. Despite substantial progress in identification of new disease-causing genes, variants, and molecular etiologies, nearly all rare diseases have no targeted therapeutics that can address their underlying molecular causes. Base editing (BE) and prime editing (PE), two newly described iterations of CRISPR-Cas9 genome editing, represent potential therapeutic strategies that could be used to precisely, efficiently, permanently, and safely correct patients' pathogenic variants and ameliorate disease sequelae. Unlike "standard" CRISPR-Cas9 genome editing, these technologies do not rely on double-strand break (DSB) formation, thus improving safety by decreasing the likelihood of undesired insertions and deletions (indels) at the target site. Here, we provide an overview of BE and PE, including their structures, mechanisms, and differences from standard CRISPR-Cas9 genome editing. We describe several examples of the use of BE and PE to improve rare and common disease phenotypes in preclinical models and human patients, with an emphasis on in vivo editing efficacy, safety, and delivery method. We also discuss recently developed delivery methods for these technologies that may be used in future clinical settings.
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Analysis of conventional and alternative CRISPR/Cas9 genome editing to enhance a single-base pair knock-in mutation.
BACKGROUND: The use of CRISPR/Cas9 technologies in generating single-base pair knock-in mutations has recently exploded in the number of methods available. However, with the growing expansion of new technologies, it can be difficult to determine the best method for genome editing. RESULTS: In this study, we evaluated a number of CRISPR/Cas9 approaches for deriving cell lines with knock-in base pair edits to create a phosphorylation mutation and provide a breakdown of editing efficiencies and suggestions for improvement. Overall, our studies suggest that using pre-formed ribonucleoprotein (RNP) complexes is a reliable editing method to generate homozygous single-base pair mutations. We also show that antibiotic selection coupled homologous recombination is an efficient tool for generating highly specific heterozygous mutations. CONCLUSION: The methods and/or combination of methods outlined in this study can be used to help other researchers with similar goals in single-base pair genome editing.
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Gene editing and multi-omics approaches to study early embryogenesis in cattle.
Advancements in multi-omics profiling and targeted molecular functional tools have led to significant progress in our understanding of early embryonic development in mammals. This is very relevant in the beef and dairy industries for exploring the etiology of early embryo loss. This review highlights how state-of-the-art single-cell and integrative low-input omics technologies, including single-cell RNA-seq, ATAC-seq, metabolomics, and proteomics, have uncovered complex developmental dynamics during the first week of bovine embryogenesis. This review also provides a concurrent overview of the application of functional tools, including CRISPR-Cas9, RNA editing, base editing, and Trim-Away, in the analysis of critical genes/proteins during the various stages of early embryo development in cattle. The integration of high-dimensional molecular profiling with targeted manipulations enables researchers to analyze key developmental events with unparalleled resolution. It is evident that these approaches provide a more comprehensive mechanistic depiction of early embryogenesis, thereby informing translational efforts in improving bovine fertility.
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