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Genome Editing in Mouse and Rat by Electroporation.
Many genome-edited mouse and rat strains have been produced using engineered endonucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Especially, CRISPR-Cas9 is powerful tool that can be easy, rapid, and high-efficiency-produced new genome-edited strains. Furthermore, new technique, Technique for Animal Knockout system by Electroporation (TAKE), efficiently accelerate production of new strains by direct nuclease introduction into intact embryos using electroporation. This chapter presents a latest technical information in the production of genome-edited mouse and rat by TAKE method.
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CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1-5) or deletion (1-4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.
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Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.
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Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.
Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.
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Lipid and polymer mediated CRISPR/Cas9 gene editing.
A clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) system is the most widely used tool for gene editing. Due to the advantages of simple operation, high efficiency and high specificity, it has been used in various fields. In this review, we introduce the latest research in the delivery technology of CRISPR/Cas9 based on lipids and polymers, which are synthesized through chemical methods. Many successful examples and methods are discussed. In addition, we discuss the potential of these delivery systems used for CRISPR/Cas9 in clinical applications in the future.
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The promise and challenge of therapeutic genome editing.
Genome editing, which involves the precise manipulation of cellular DNA sequences to alter cell fates and organism traits, has the potential to both improve our understanding of human genetics and cure genetic disease. Here I discuss the scientific, technical and ethical aspects of using CRISPR (clustered regularly interspaced short palindromic repeats) technology for therapeutic applications in humans, focusing on specific examples that highlight both opportunities and challenges. Genome editing is-or will soon be-in the clinic for several diseases, with more applications under development. The rapid pace of the field demands active efforts to ensure that this breakthrough technology is used responsibly to treat, cure and prevent genetic disease.
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[Development of CRISPR technology and its application in bone and cartilage tissue engineering].
The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.
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Cell-Specific Control of Mammalian Gene Expression Using DNA Repair Inducible Ribozyme Switches.
The ability to control gene expression is vital for elucidating gene functions and developing next-generation therapeutics. Current techniques are challenged by the lack of cell-specific control designs or immunogenicity risk from foreign proteins. We develop a DNA repair inducible ribozyme switch that enables cell-specific control of gene expression in cells and in vivo. This strategy designs plasmids with a DNA lesion (8-oxoG and O6-MeG) site-specifically installed within the ribozyme encoding region, generating active hammerhead ribozyme for mRNA degradation due to transcriptional mutagenesis, whereas DNA repair yields a single-base mismatch in the ribozyme to abrogate its activity. This strategy is demonstrated to allow specific control of gene expression in cancer cells with overexpressed DNA repair enzymes such as MutY DNA glycosylase and O6-methylguanine-DNA-methyltransferases. It also shows the capability of conditionally regulating the expression of different proteins for signal reporting and gene editing, enabling DNA repair monitoring and targeted gene therapy in cancer cells. This strategy is demonstrated using the inducible CRISPR/Cas9 system for in vivo editing of oncogenic Polo-like kinase 1 in a mouse model, resulting in significant tumor growth suppression. The DNA repair inducible ribozyme switch may provide a compact system for cell-specific gene expression control toward precise gene therapy.
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Exploring the Function of Gene Promoter Regulatory Elements Using CRISPR Tools.
Gene promoters serve as pivotal regulators of transcription, orchestrating the initiation, rate, and specificity of gene expression, resulting in cellular diversity found among distinct cell types within multicellular organisms. Identification of the sequence and function of promoters' regulatory elements and their complex interaction with transcription factors, enhancers, silencers, and insulators is fundamental to coordinated transcriptional processes within cells. Identifying these regulatory elements and scrutinizing their functions and interactions through the use of synthetic promoters can pave the way for researchers in various fields ranging from uncovering the origins of diseases associated with promoter mutations to harnessing these regulatory components in biotechnological applications.In this chapter, we describe the manipulation of regulatory elements within promoters, with a specific focus on the use of CRISPR technology on enhancers and silencer elements of the Ovalbumin gene promoter. We explain and discuss processes for the deletion of/interference with regulatory elements within the promoter, employing CRISPR-based approaches. Furthermore, we demonstrate that a CRISPR/Cas-manipulated promoter can activate gene transcription in cell types where it is normally inactive. This confirms that CRISPR technology can be effectively used to engineer synthetic promoters with desired characteristics, such as inducibility, tissue-specificity, or enhanced transcriptional strength. Such an approach provides valuable insights into the mechanisms and dynamics of gene expression, thereby offering new opportunities in the fields of biotechnology and medicine.
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Efficient genome editing in plants using a CRISPR/Cas system.
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