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Cas9 immunity creates challenges for CRISPR gene editing therapies.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a genome-editing technology, that utilizes archaeal and bacterial Cas9 nucleases to introduce double-stranded breaks in DNA at targeted sites. These breaks can be used to remove, replace, or add pieces of DNA. While not the first genome editor, CRISPR-Cas9 is efficient and cost-effective because cutting is guided by a strand of RNA rather than a protein. The potential uses in health care are plentiful, from disrupting dominant genes that cause cancer to repairing mutated genes that cause genetic diseases, such as muscular dystrophy. Therapeutic approaches based on this technology fill the preclinical pipeline, and rely on the use of viral vectors to deliver the Cas9 gene and guide RNA to a gene of interest. However, concerns regarding the safety and efficacy of CRISPR-Cas9 use in gene therapy remain. A pre-print released prior to peer review has recently underlined the question of whether immunological responses to Cas9 may negatively impact its clinical use. Here we discuss the implications of this finding for the application of CRISPR/Cas in gene therapy.
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Recent advances in CRISPR technologies for biomedical applications and disease treatment.
Clustered regularly interspaced short palindromic repeats (CRISPR), a precise genome-editing platform, has revolutionized genetic engineering. The highly selective and programmable characteristics of CRISPR have enabled its broad application. Recent advances in CRISPR biotechnology have promoted its widespread use in various biomedical fields, such as CRISPR screening. Ongoing preclinical and clinical trials will help determine the full capabilities of CRISPR in disease therapy. The efficacy of CRISPR-based applications is fundamentally dependent on optimized delivery strategies. Multiple approaches for delivering the CRISPR components have been investigated, primarily relying on physical, viral and non-viral delivery methods. Each method has specific strengths and limitations. Notably, non-viral delivery systems hold significant promise for CRISPR applications, offering distinct advantages such as transient expression, low immunogenicity, and fewer size constraints for cargo delivery. Various biomaterials demonstrate strong potential for delivering CRISPR components to achieve gene editing, including inorganic nanoparticles, polymeric nanoparticles, lipid nanoparticles, metal-organic frameworks, DNA origami, and virus-like particles. Therefore, this review provides an overview of recent research on the molecular principles of CRISPR and its biomedical applications. Recent advances in CRISPR-based treatments and their delivery vectors are also reviewed. Finally, the challenges faced by CRISPR technology are discussed.
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Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans.
The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline.
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A Single-Molecule View of Genome Editing Proteins: Biophysical Mechanisms for TALEs and CRISPR/Cas9.
Exciting new advances in genome engineering have unlocked the potential to radically alter the treatment of human disease. In this review, we discuss the application of single-molecule techniques to uncover the mechanisms behind two premier classes of genome editing proteins: transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas). These technologies have facilitated a striking number of gene editing applications in a variety of organisms; however, we are only beginning to understand the molecular mechanisms governing the DNA editing properties of these systems. Here, we discuss the DNA search and recognition process for TALEs and Cas9 that have been revealed by recent single-molecule experiments.
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Livestock Gene Editing by One-step Embryo Manipulation.
The breakthrough and rapid advance of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has enabled the efficient generation of gene-edited animals by one-step embryo manipulation. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 delivery to the livestock embryos has been typically achieved by intracytoplasmic microinjection; however, recent studies show that electroporation may be a reliable, efficient, and practical method for CRISPR/Cas9 delivery. The source of embryos used to generate gene-edited animals varies from in vivo to in vitro produced, depending mostly on the species of interest. In addition, different Cas9 and gRNA reagents can be used for embryo editing, ranging from Cas9-coding plasmid or messenger RNA to Cas9 recombinant protein, which can be combined with in vitro transcribed or synthetic guide RNAs. Mosaicism is reported as one of the main problems with generation of animals by embryo editing. On the other hand, off-target mutations are rarely found in livestock derived from one-step editing. In this review, we discussed these and other aspects of generating gene-edited animals by single-step embryo manipulation.
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Making point mutations in Escherichia coli BL21 genome using the CRISPR-Cas9 system.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system is an efficient and rapid tool for genome editing. However, its utilization in bacteria suffers challenges such as the risk of repeated recognition and cutting by Cas9. Here we established a two-step genome editing strategy using the Streptococcus pyogenes CRISPR-Cas9 system to achieve a clean mutation with only the target sites into the Escherichia coli genome. This strategy can avoid the risk of repeated cutting by guide RNA (gRNA)/Cas9 without altering the protospacer-adjacent motif or inserting additional silent mutations into the genome. The principles and approaches we developed in this study can be applied to modify coding and non-coding sequences in essential and non-essential genes and can also be used for precise genome editing in other microorganisms.
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Genome Editing for the Study of Cardiovascular Diseases.
PURPOSE OF REVIEW: The opportunities afforded through the recent advent of genome-editing technologies have allowed investigators to more easily study a number of diseases. The advantages and limitations of the most prominent genome-editing technologies are described in this review, along with potential applications specifically focused on cardiovascular diseases. RECENT FINDINGS: The recent genome-editing tools using programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have rapidly been adapted to manipulate genes in a variety of cellular and animal models. A number of recent cardiovascular disease-related publications report cases in which specific mutations are introduced into disease models for functional characterization and for testing of therapeutic strategies. Recent advances in genome-editing technologies offer new approaches to understand and treat diseases. Here, we discuss genome editing strategies to easily characterize naturally occurring mutations and offer strategies with potential clinical relevance.
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Creating cell and animal models of human disease by genome editing using CRISPR/Cas9.
A set of unique sequences in bacterial genomes, responsible for protecting bacteria against bacteriophages, has recently been used for the genetic manipulation of specific points in the genome. These systems consist of one RNA component and one enzyme component, known as CRISPR ("clustered regularly interspaced short palindromic repeats") and Cas9, respectively. The present review focuses on the applications of CRISPR/Cas9 technology in the development of cellular and animal models of human disease. Making a desired genetic alteration depends on the design of RNA molecules that guide endonucleases to a favorable genomic location. With the discovery of CRISPR/Cas9 technology, researchers are able to achieve higher levels of accuracy because of its advantages over alternative methods for editing genome, including a simple design, a high targeting efficiency and the ability to create simultaneous alterations in multiple sequences. These factors allow the researchers to apply this technology to creating cellular and animal models of human diseases by knock-in, knock-out and Indel mutation strategies, such as for Huntington's disease, cardiovascular disorders and cancers. Optimized CRISPR/Cas9 technology will facilitate access to valuable novel cellular and animal genetic models with respect to the development of innovative drug discovery and gene therapy.
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Engineering guide RNA to reduce the off-target effects of CRISPR.
As versatile and robust genome editing tools, clustered regularly interspaced short palindromic repeats (CRISPR) technologies have been broadly used in basic research, biotechnology, and therapeutic development. Off-target mutagenesis by CRISPR systems has been demonstrated, and various methods have been developed to markedly increase their specificity. In this review, we highlight the efforts of producing and modifying guide RNA (gRNA) to minimize off-target activities, including sequence and structure design, tuning expression and chemical modification. The modalities of gRNA engineering can be applied across CRISPR systems. In conjunction with CRISPR protein effectors, the engineered gRNA enables efficient and precise genome editing.
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Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.
The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.
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