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CRISPR heavyweights battle in US patent court.
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Large DNA fragment knock-in and sequential gene editing in Plasmodium falciparum: a preliminary study using suicide-rescue-based CRISPR/Cas9 system.
CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce "marker-free" genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.
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Generation of Rabbit Models by Gene Editing Nucleases.
Due to the lack of germline transmitting pluripotent stem cells (PSCs) cell lines and the extreme difficulty of somatic cell nuclear transfer (SCNT) in rabbit, the gene targeting technology in rabbit was lagging far behind those in rodents and in farm animals. As a result, the development and application of genetically engineered rabbit model are much limited. With the advent of gene editing nucleases, including ZFN, TALEN, and CRISPR/Cas9, it is now possible to produce gene targeting (i.e., knockout, knockin) rabbits with high success rates. In this chapter, we describe a comprehensive, step-by-step protocol for rabbit genome editing based on gene editing nucleases with specific emphasis of CRISPR/Cas9.
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Targeted Transgenic Mice Using CRISPR /Cas9 Technology.
CRISPR /Cas9 is a powerful technology that has transformed gene editing of mammalian genomes, being faster and more cost-effective than standard gene targeting techniques. In this chapter, we provide a step-by-step protocol to obtain Knock-Out (KO ) or Knock-In (KI ) mouse models using CRISPR /Cas9 technology. Detailed instructions for the design of single guide RNAs (sgRNA ) for KO approaches and single-strand oligonucleotide (ssODN ) matrix for generation of KI animals are included. We also describe two independent CRISPR /Cas9 delivery methods to produce gene-edited animals starting from zygote-stage embryos, based either on cytoplasmic injection or electroporation.
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CRISPR-Cas9: A fascinating journey from bacterial immune system to human gene editing.
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system has been discovered as an adaptive-immune system in prokaryotes. Microbes like bacteria and archaea use CRISPR-Cas9 as a part of their defense mechanism to ward off the virus and cleave their DNA. Over the past decades, researchers have identified that this simple CRISPR-Cas9 system of bacteria can be utilized to cut any DNA. It is also possible to make precise editing in the genome of almost any organism. This discovery has revolutionized the CRISPR-Cas9 tools and made it one of the most precise gene editing technology known till date. The simple, versatile and programmable nature of CRISPR-Cas9 system 5wthat contains a single guide RNA and Cas9 enzyme, made it an attractive choice for genome editing application. Scientists in the field of molecular biology, genetics and medicine extensively use this transformative technology to study gene regulation and also for treatment of several incurable genetic diseases. Today, CRISPR-Cas9 is the most powerful breakthrough of the century for its immense potential to modulate gene expression in living cells and its application to medicine and human health. Recently, ethical challenges associated with the application of this technology to human health become a hot debate in the scientific community. In this chapter the brief history of development of CRISPR-Cas9 tools and its immense application potential have been discussed.
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Application of Cas9-Based Gene Editing to Engineering of Nonribosomal Peptide Synthetases.
Engineering of nonribosomal peptide synthetases (NRPSs) could transform the production of bioactive natural product derivatives. A number of recent reports have described the engineering of NRPSs without marked reductions in yield. Comparative analysis of evolutionarily related NRPSs can provide insights regarding permissive fusion sites for engineering where recombination may occur during evolutionary processes. Studies involving engineering of NRPSs using these recombination sites showed that they have great potential. Moreover, we highlight recent advances in engineering of NRPSs using CRISPR-associated protein 9 (Cas9)-based gene editing technology. The use of Cas9 facilitates the editing of even larger biosynthetic gene clusters (BGCs) close to or over 100 kb in size by precisely targeting and digesting DNA sequences at specific sites. This technology combined with growing understanding of potential fusion sites from large-scale informatics analyses will accelerate the scalable exploration of engineered NRPS assembly lines to obtain bioactive natural product derivatives in high yields.
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New CRISPR-Cas9 vectors for genetic modifications of Bacillus species.
Genetic manipulation is a fundamental procedure for the study of gene and operon functions and new characteristics acquisition. Modern CRISPR-Cas technology allows genome editing more precisely and increases the efficiency of transferring mutations in a variety of hard to manipulate organisms. Here, we describe new CRISPR-Cas vectors for genetic modifications in bacillary species. Our plasmids are single CRISPR-Cas plasmids comprising all components for genome editing and should be functional in a broad host range. They are highly efficient (up to 97%) and precise. The employment and delivery of these plasmids to bacillary strains can be easily achieved by conjugation from Escherichia coli. During our research we also demonstrated the absence of compatibility between CRISPR-Cas system and non-homologous end joining in Bacillus subtilis.
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Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.
The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.
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CRISPR diagnostics: Underappreciated uses in perinatology.
CRISPR-based therapeutics have the potential to revolutionize the treatment of hereditary diseases, but current efforts to translate research to the bedside face significant technical, regulatory, and ethical hurdles. In this article, we discuss an underappreciated application of CRISPR: diagnostic testing, and argue that: (1) CRISPR diagnostics are poised to disrupt diagnostic practices including perinatal screening and (2) since CRISPR diagnostics pose minimal technical, regulatory and ethical hurdles (unlike CRISPR therapeutic uses) they are likely to be clinically relevant before CRISPR-based therapies, and thus warrant medical community's attention.
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Efficient Gene Editing in Fish Primary Germline Stem Cells.
Genome editing by the CRISPR/Cas9 system is widely used for production of gene-modified animals, including fish. However, efficient gene editing in fish cultured cells, in particular germline stem cells (GSCs), is challenging, likely due to the difficulty in transfecting these cells. The ricefield eel (Monopterus albus), a sequential hermaphroditic species, is a freshwater fish of significant economic value in China. In this work, we report a simple method that can achieve high gene editing efficiency in primary GSCs of fish species, including ricefield eel. High transfection efficiency (50-95%) is achieved in fish GSCs by using a microchannel-based cell transfection system. Gene editing efficiency of up to 60% in primary ricefield eel GSCs is achieved using an integrated CRISPR/Cas9 vector strategy. High gene editing efficiency is also achieved in gibel carp (Carassius gibelio) GSCs and the medaka spermatogonial stem cell line SG3, demonstrating the general applicability of our method. Our data suggest that efficient transfection is key to high gene editing efficiency in fish cultured GSCs. This study establishes an efficient and reliable gene editing system for fish GSCs, which may facilitate the creation of new germplasm of genetically difficult-to-breed fish by combining GSC transplantation with gene editing techniques.
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