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Advances in CRISPR Delivery Methods: Perspectives and Challenges.
With the advent of new genome editing technologies and the emphasis placed on their optimization, the genetic and phenotypic correction of a plethora of diseases sit on the horizon. Ideally, genome editing approaches would provide long-term solutions through permanent disease correction instead of simply treating patients symptomatically. Although various editing machinery options exist, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein) editing technique has emerged as the most popular due to its high editing efficiency, simplicity, and affordability. However, while CRISPR technology is gradually being perfected, optimization is futile without accessible, effective, and safe delivery to the desired cell or tissue. Therefore, it is important that scientists simultaneously focus on inventing and improving delivery modalities for editing machinery as well. In this review, we will discuss the critical details of viral and nonviral delivery systems, including payload, immunogenicity, efficacy in delivery, clinical application, and future directions.
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Genomic Determinants of THAP11/ZNF143/HCFC1 Complex Recruitment to Chromatin.
The THAP11 and ZNF143 transcription factors recognize overlapping DNA sequences and are reported to exhibit signs of both competitive and cooperative binding. HCFC1 serves as a scaffold protein, bridging interactions between transcription factors, including THAP11 and ZNF143, and transcriptional coregulators. The exact mechanism of how DNA sequences guide the recruitment of the THAP11/ZNF143/HCFC1 complex to chromatin is still controversial. In this study, we use chromosomally integrated synthetic constructs and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated approaches in intact cells to elucidate the role of the DNA sequence in the recruitment of this complex and to establish its biological relevance. We show that the ACTACA submotif, shared by both THAP11 and ZNF143, directs the recruitment of THAP11 and HCFC1 to ZNF143-occupied loci. Importantly, its position, spacing, and orientation relative to the ZNF143 core motif are critical for this action. CRISPR-Cas9-mediated alterations of the ACTACA submotif at endogenous promoters recapitulated results obtained with synthetic constructs and resulted in altered gene transcription and histone modifications at targeted promoters. Our in vivo approaches provide strong evidence for the molecular role of the ACTACA submotif in THAP11, ZNF143, and HCFC1 cooperative recruitment to chromatin and its biological role in target gene expression.
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The application of gene silencing in proteomics: from laboratory to clinic.
Since the completion of genome sequencing, gene silencing technologies have emerged as powerful tools to study gene functions in various biological processes, both in vivo and in vitro. Moreover, they have also been proposed as therapeutic agents to inhibit selected genes in a variety of pathological conditions, such as cancer, neurodegenerative, and cardiovascular diseases. Area covered: This review summarizes the mechanisms of action and applications of genome editing tools, from RNA interference to clustered regularly interspaced short palindromic repeats-based systems, in research and in clinics. We describe their essential role in high-throughput genetic screens and, in particular, in functional proteomics studies, to identify diagnostic markers and therapeutic targets. Indeed, gene silencing and proteomics have been extensively integrated to study global proteome changes, posttranslational modifications, and protein-protein interactions. Expert commentary: Functional proteomics approaches that leverage gene silencing tools have been successfully applied to examine the role of several genes in various contexts, leading to a deeper knowledge of biological pathways and disease mechanisms. Recent developments of gene silencing tools have improved their performance, also in terms of off-targets effects reduction, paving the way for a wider therapeutic application of these systems.
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CRISPR/Cas9 and genome editing in Drosophila.
Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.
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Reactivation of FMR1 by CRISPR/Cas9-Mediated Deletion of the Expanded CGG-Repeat of the Fragile X Chromosome.
Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle, we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies. The reactivated cells produced FMRP and exhibited a decline in DNA methylation at the FMR1 locus. These data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation.
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A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing.
Gene editing is a versatile technique in biomedicine that promotes fundamental research as well as clinical therapy. The development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a genome editing machinery has accelerated the application of gene editing. However, the delivery of CRISPR components often suffers when using conventional transfection methods, such as viral transduction and chemical vectors, due to limited packaging size and inefficiency toward certain cell types. In this review, we discuss physical transfection methods for CRISPR gene editing which can overcome these limitations. We outline different types of physical transfection methods, highlight novel techniques to deliver CRISPR components, and emphasize the role of micro and nanotechnology to improve transfection performance. We present our perspectives on the limitations of current technology and provide insights on the future developments of physical transfection methods.
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CRISPR-Cas systems: Challenges and future prospects.
The advancement gained over the past couple of decades in clustered regularly interspaced short palindromic repeats and CRISPR associated proteins (CRISPR-Cas) systems have revolutionized the field of synthetic biology, therapeutics, diagnostics and metabolic engineering. The technique has enabled the process of genome editing to be very precise, rapid, cost-effective and highly efficient which were the downfalls for the previously debuted zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) technologies. However, despite its great potential, challenges including off-target activity, method of delivery, ethical and regulatory issues still remain unresolved for the CRISPR-Cas systems. In this chapter, we present and point out the obstacles faced in implementation of the CRISPR-Cas system along with its future prospects.
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pHNRhCas9NG, single expression cassette-based dual-component dual-transcription unit CRISPR/Cas9 system for plant genome editing.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing (GEd) technology has revolutionized plant science, facilitating gene function studies and crop improvement. Despite its success, plant-specific CRISPR/Cas9 systems require further optimization. This study aims to boost plant GEd efficiency by revamping the CRISPR/Cas9 system. We addressed large fragment deletions in T-DNA (transfer DNA) postgenomic insertion by developing a binary expression vector, pHNR, which maintains T-DNA integrity using protective sequences. We discovered an artificial promoter, P35SIC47, effective in tobacco, Arabidopsis, and tomato transformation, and designed a dual-component dual-transcription unit CRISPR/Cas9 system (DDS) with optimal gene expression at a poly(A) length of ~150 base pairs. Enhancing the poly(A) tail length of Cas9 mRNA significantly boosted plant GEd efficiency. We also identified compatible hCas9 versions through transitory expression in tobacco leaves. Utilizing pHNRhCas9NG, we efficiently knocked out ten genes in tomato, achieving almost 100% gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.
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High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.
Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.
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Efficient CRISPR/Cas9-based genome editing in carrot cells.
The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116-119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop.
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