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Rapid Control of Genome Editing in Human Cells by Chemical-Inducible CRISPR-Cas Systems.
Genome editing using programmable DNA endonucleases enables the engineering of eukaryotic cells and living organisms with desirable properties or traits. Among the various molecular scissors that have been developed to date, the most versatile and easy-to-use family of nucleases derives from CRISPR-Cas, which exists naturally as an adaptive immune system in bacteria. Recent advances in the CRISPR-Cas technology have expanded our ability to manipulate complex genomes for myriad biomedical and biotechnological applications. Some of these applications are time-sensitive or demand high spatial precision. Here, we describe the use of an inducible CRISPR-Cas9 system, termed iCas, which we have developed to enable rapid and tight control of genome editing in mammalian cells. The iCas system can be switched on or off as desired through the introduction or removal of the small molecule tamoxifen or its related analogs such as 4-hydroxytamoxifen (4-HT).
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Toward In Vivo Gene Therapy Using CRISPR.
CRISPR, a revolutionizing technology allowing researchers to navigate in and edit the genome, is moving on the fast track toward clinical use for ex vivo correction of disease-causing mutations in stem cells. As we await the first trials utilizing ex vivo CRISPR editing, implementation of CRISPR-based gene editing as an in vivo treatment directly in patients still remains an ultimate challenge. However, quickly accumulating evidence has provided proof-of-concept for efficacious editing in vivo. Attempts to edit genes directly in animals have largely relied on classical vector systems based on virus-based delivery of gene cassettes encoding the Cas9 endonuclease and single guide RNA, the key components of the CRISPR system. However, whereas persistent gene expression has been the primary goal of gene therapy for decades, things may be different in the case of CRISPR delivery. Is short-term presence of the CRISPR components perhaps sufficient for efficacy and ideal for safety?-and are strategies needed for restricting immune recognition of the bacteria-derived editing tool? Here, while answers to these questions still blow in the wind, we review prominent examples of genome editing with focus on targeting of genes with CRISPR in liver, muscles, and eyes of the mouse.
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Gene Editing in 3D Cultured Nephron Progenitor Cell Lines.
During kidney development, Six2+ nephron progenitor cells (NPCs) generate nephrons, the functional units of the kidney. Isolating and expanding NPCs in vitro have enabled wide applications in both basic and translational research. Here we describe the methods to derive NPC lines from mouse embryonic kidneys in a 3D culture format as well as gene editing in the NPC lines.
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[The new generation tool for CRISPR genome editing: CRISPR/Cpf1].
Almost all archaea and many bacteria achieve adaptive immunity through a diverse set of CRISPR-Cas systems. From 2012, Cas9 has been harnessed by thousands of laboratories for genome editing applications in a variety of experimental model systems. Cas9 is driving innovative applications from basic biology to biotechnology and medicine. However, numerous challenges still remain, such as limited targeting range, toxicity, and potential for off-target mutagenesis. Cpf1 represents a class 2/type V CRISPR RNA-guided endonuclease that is distinct from the type II CRISPR Cas9 nuclease. Future exploration of strategies using Cpf1 is expected to bring solutions for some of the biggest challenges facing genome editing. Here, we review the development and applications of Cpf1 for a variety of research or applications, and highlight challenges as well as future directions.
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Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos.
Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.
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Genetics in orthopaedic practice.
DNA holds genetic information in the nucleus of eukaryotic cells; and has three different functions: replication, storage of hereditary information, and regulation of cell division. Most studies described the association of single nucleotide polymorphism (SNP) to common orthopaedics diseases and the susceptibility to develop musculoskeletal injuries. Several mutations are associated with osteoporosis, musculoskeletal ailments and other musculoskeletal deformity and conditions. Several strategies, including gene therapy and tissue engineering with mesenchymal stem cells (MSC), have been proposed to enhance healing of musculoskeletal tissues. Furthermore, a recent technique has revolutionized gene editing: clustered regulatory interspaced short palindromic repeat (CRISPR) technology is characterized by simplicity in target design, affordability, versatility, and high efficiency, but needs more studies to become the preferred platform for genome editing. Predictive genomics DNA profiling allows to understand which genetic advantage, if any, may be exploited, and why a given rehabilitation protocol can be more effective in some individual than others. In conclusion, a better understanding of the genetic influence on the function of the musculoskeletal system and healing of its ailments is needed to plan and develop patient specific management strategies.
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CRISPR Libraries and Screening.
CRISPR-Cas9 technology has revolutionized large-scale functional genomic screening in mammalian cell-culture systems. Due in part to optimized lentiviral delivery vectors; it is now possible to perform CRISPR-Cas9 screens in animals in order to study biological processes in the context of a whole organism and within more physiologically relevant environment. This chapter focuses primarily on mouse models of human cancers; viral vectors used for simultaneous tumor initiation and genome editing and sgRNA library design considerations. Experience with direct and indirect in vivo RNAi screens in the literature is also discussed in order to highlight the challenges of delivering diverse libraries of small RNAs in vivo.
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In Silico Meets In Vivo: Towards Computational CRISPR-Based sgRNA Design.
CRISPR-based genome editing has been widely implemented in various cell types. In silico single guide RNA (sgRNA) design is a key step for successful gene editing using the CRISPR system, and continuing efforts are aimed at refining in silico sgRNA design with high on-target efficacy and reduced off-target effects. Many sgRNA design tools are available, but careful assessments of their application scenarios and performance benchmarks across different types of genome-editing data are needed. Efficient in silico models can be built that integrate current heterogeneous genome-editing data to derive unbiased sgRNA design rules and identify key features for improving sgRNA design. Comprehensive evaluation of on-target and off-target effects of sgRNA will allow more precise genome editing and gene therapies using the CRISPR system.
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mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.
Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly) manipulated at multiple loci with extremely high efficiency.
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Manipulating the Destiny of Wild Populations Using CRISPR.
Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches.
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