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Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii.
Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.
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Building CRISPR Gene Therapies for the Central Nervous System: A Review.
IMPORTANCE: Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) holds the promise to arrest or cure monogenic disease if it can be determined which genetic change to create without inducing unintended cellular dysfunction and how to deliver this technology to the target organ reliably and safely. Clinical trials for blood and liver disorders, for which delivery of CRISPR is not limiting, show promise, yet no trials have begun for central nervous system (CNS) indications. OBSERVATIONS: The CNS is arguably the most challenging target given its innate exclusion of large molecules and its defenses against bacterial invasion (from which CRISPR originates). Herein, the types of CRISPR editing (DNA cutting, base editing, and templated repair) and how these are applied to different genetic variants are summarized. The challenges of delivering genome editors to the CNS, including the viral and nonviral delivery vehicles that may ultimately circumvent these challenges, are discussed. Also, ways to minimize the potential in vivo genotoxic effects of genome editors through delivery vehicle design and preclinical off-target testing are considered. The ethical considerations of germline editing, a potential off-target outcome of any gene editing therapy, are explored. The unique regulatory challenges of a human-specific therapy that cannot be derisked solely in animal models are also discussed. CONCLUSIONS AND RELEVANCE: An understanding of both the potential benefits and challenges of CRISPR gene therapy better informs the scientific, clinical, regulatory, and timeline considerations of developing CRISPR gene therapy for neurologic diseases.
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Bringing CRISPR to the Cinema.
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Increasing the targeting scope and efficiency of base editing with Proxy-BE strategy.
Base editors (BEs) are widely used in precise gene editing due to their simplicity and versatility. However, their efficiencies are hindered by various obstacles. Considering the chromatin microenvironment as a possible obstacle, here, we demonstrate a further development of the proxy-clustered regularly interspaced short palindromic repeats strategy, termed Proxy-BE, to increase gene editing efficiency. Specifically, a nuclease-dead Cas9 (dCas9) was bound to the sequence about 20-30 base pair away from the target site, potentially improving access to the DNA and, thus, providing a better editing microenvironment for base editors. Our findings confirm that nuclease-dead Streptococcus pyogenes Cas9 can assist the base editors SaKKH-BE3 and dCpf1-BE to double their canonical base editing efficiency. This work provides a new approach to enhance base editing, extending its scope for biological research and gene therapy.
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Now on Course, CRISPR J.
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Taking CRISPR to New Heights.
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Highly efficient RNA-guided base editing in mouse embryos.
Base editors (BEs) composed of a cytidine deaminase fused to CRISPR-Cas9 convert cytidine to uridine, leading to single-base-pair substitutions in eukaryotic cells. We delivered BE mRNA or ribonucleoproteins targeting the Dmd or Tyr gene via electroporation or microinjection into mouse zygotes. F0 mice showed nonsense mutations with an efficiency of 44-57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations.
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CRISPR-Cas tools to study gene function in cytokinesis.
Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Finally, we address the need for gene editing to study cytokinesis in more physiologically relevant contexts. Therefore, this Review provides a roadmap for gene editing to be used in the study of cytokinesis and other cellular processes.
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CRISPR Milestones for Sustainable Agriculture and Forestry.
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Advances in genome editing for genetic hearing loss.
According to the World Health Organization, hearing loss affects over 466 million people worldwide and is the most common human sensory impairment. It is estimated that genetic factors contribute to the causation of approximately 50% of congenital hearing loss. Yet, curative approaches to reversing or preventing genetic hearing impairment are still limited. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) systems enable programmable and targeted gene editing in highly versatile manners and offer new gene therapy strategies for genetic hearing loss. Here, we summarize the most common deafness-associated genes, illustrate recent strategies undertaken by using CRISPR-Cas9 systems for targeted gene editing and further compare the CRISPR strategies to non-CRISPR gene therapies. We also examine the merits of different vehicles and delivery forms of genome editing agents. Lastly, we describe the development of animal models that could facilitate the eventual clinical applications of the CRISPR technology to the treatment of genetic hearing diseases.
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