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Genome-editing tools for stem cell biology.
Human pluripotent stem cells provide a versatile platform for regenerative studies, drug testing and disease modeling. That the expression of only four transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), is sufficient for generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells has revolutionized the field and also highlighted the importance of OKSM as targets for genome editing. A number of novel genome-editing systems have been developed recently. In this review, we focus on successful applications of several such systems for generation of iPSCs. In particular, we discuss genome-editing systems based on zinc-finger fusion proteins (ZFs), transcription activator-like effectors (TALEs) and an RNA-guided DNA-specific nuclease, Cas9, derived from the bacterial defense system against viruses that utilizes clustered regularly interspaced short palindromic repeats (CRISPR).
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CRISPR multitargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences.
Genome engineering has been revolutionized by the discovery of clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated system genes (Cas) in bacteria. The type IIB Streptococcus pyogenes CRISPR/Cas9 system functions in many species and additional types of CRISPR/Cas systems are under development. In the type II system, expression of CRISPR single guide RNA (sgRNA) targeting a defined sequence and Cas9 generates a sequence-specific nuclease inducing small deletions or insertions. Moreover, knock-in of large DNA inserts has been shown at the sites targeted by sgRNAs and Cas9. Several tools are available for designing sgRNAs that target unique locations in the genome. However, the ability to find sgRNA targets common to several similar sequences or, by contrast, unique to each of these sequences, would also be advantageous. To provide such a tool for several types of CRISPR/Cas system and many species, we developed the CRISPR MultiTargeter software. Similar DNA sequences in question are duplicated genes and sets of exons of different transcripts of a gene. Thus, we implemented a basic sgRNA target search of input sequences for single-sgRNA and two-sgRNA/Cas9 nickase targeting, as well as common and unique sgRNA target searches in 1) a set of input sequences; 2) a set of similar genes or transcripts; or 3) transcripts a single gene. We demonstrate potential uses of the program by identifying unique isoform-specific sgRNA sites in 71% of zebrafish alternative transcripts and common sgRNA target sites in approximately 40% of zebrafish duplicated gene pairs. The design of unique targets in alternative exons is helpful because it will facilitate functional genomic studies of transcript isoforms. Similarly, its application to duplicated genes may simplify multi-gene mutational targeting experiments. Overall, this program provides a unique interface that will enhance use of CRISPR/Cas technology.
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Review of CRISPR/Cas9 sgRNA Design Tools.
The adaptive immunity system in bacteria and archaea, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-associate (CRISPR/Cas), has been adapted as a powerful gene editing tool and got a broad application in genome research field due to its ease of use and cost-effectiveness. The performance of CRISPR/Cas relies on well-designed single-guide RNA (sgRNA), so a lot of bioinformatic tools have been developed to assist the design of highly active and specific sgRNA. These tools vary in design specifications, parameters, genomes and so on. To help researchers to choose their proper tools, we reviewed various sgRNA design tools, mainly focusing on their on-target efficiency prediction model and off-target detection algorithm.
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Therapeutic applications of CRISPR/Cas9 gene editing technology for the treatment of ocular diseases.
Ocular diseases are a highly heterogeneous group of phenotypes, caused by a spectrum of genetic variants and environmental factors that exhibit diverse clinical symptoms. As a result of its anatomical location, structure and immune privilege, the eye is an ideal system to assess and validate novel genetic therapies. Advances in genome editing have revolutionized the field of biomedical science, enabling researchers to understand the biology behind disease mechanisms and allow the treatment of several health conditions, including ocular pathologies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing facilitates efficient and specific genetic modifications in the nucleic acid sequence, resulting in permanent changes at the genomic level. This approach has advantages over other treatment strategies and is promising for the treatment of various genetic and non-genetic ocular conditions. This review provides an overview of the CRISPR/CRISPR-associated protein 9 (Cas9) system and summarizes recent advances in the therapeutic application of CRISPR/Cas9 for the treatment of various ocular pathologies, as well as future challenges.
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A novel CCCH-type zinc finger protein SAW1 activates OsGA20ox3 to regulate gibberellin homeostasis and anther development in rice.
Male sterility is a prerequisite for hybrid seed production. The phytohormone gibberellin (GA) is involved in regulating male reproductive development, but the mechanism underlying GA homeostasis in anther development remains less understood. Here, we report the isolation and characterization of a new positive regulator of GA homeostasis, swollen anther wall 1 (SAW1), for anther development in rice (Oryza sativa L.). Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRIPSR-associated protein 9-mediated knockout of SAW1 in rice generated similar male sterile plants. SAW1 encodes a novel nucleus-localizing CCCH-tandem zinc finger protein, and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther-specific expression. In the saw1 anther, the significantly decreased OsGA20ox3 expression resulted in lower bioactive GA content, which in turn caused the lower expression of the GA-inducible anther-regulator gene OsGAMYB. Thus, our results disclose the mechanism of the SAW1-GA20ox3-GAMYB pathway in controlling rice anther development, and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.
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The application and progression of CRISPR/Cas9 technology in ophthalmological diseases.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is an adaptive immune defence system that has gradually evolved in bacteria and archaea to combat invading viruses and exogenous DNA. Advances in technology have enabled researchers to enhance their understanding of the immune process in vivo and its potential for use in genome editing. Thus far, applications of CRISPR/Cas9 genome editing technology in ophthalmology have included gene therapy for corneal dystrophy, glaucoma, congenital cataract, Leber's congenital amaurosis, retinitis pigmentosa, Usher syndrome, fundus neovascular disease, proliferative vitreoretinopathy, retinoblastoma and other eye diseases. Additionally, the combination of CRISPR/Cas9 genome editing technology with adeno-associated virus vector and inducible pluripotent stem cells provides further therapeutic avenues for the treatment of eye diseases. Nonetheless, many challenges remain in the development of clinically feasible retinal genome editing therapy. This review discusses the development, as well as mechanism of CRISPR/Cas9 and its applications and challenges in gene therapy for eye diseases.
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DeepCRISPR: optimized CRISPR guide RNA design by deep learning.
A major challenge for effective application of CRISPR systems is to accurately predict the single guide RNA (sgRNA) on-target knockout efficacy and off-target profile, which would facilitate the optimized design of sgRNAs with high sensitivity and specificity. Here we present DeepCRISPR, a comprehensive computational platform to unify sgRNA on-target and off-target site prediction into one framework with deep learning, surpassing available state-of-the-art in silico tools. In addition, DeepCRISPR fully automates the identification of sequence and epigenetic features that may affect sgRNA knockout efficacy in a data-driven manner. DeepCRISPR is available at http://www.deepcrispr.net/ .
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Genome editing in nonhuman primates: approach to generating human disease models.
Nonhuman primates (NHPs) are superior than rodents to be animal models for the study of human diseases, due to their similarities in terms of genetics, physiology, developmental biology, social behaviour and cognition. Transgenic animals have become a key tool in functional genomics to generate models for human diseases and validate new drugs. However, until now, progress in the field of transgenic NHPs has been slow because of technological limitations. Many human diseases, including neurodegenerative disorders, are caused by mutations in endogenous genes. Fortunately, recent developments in precision gene editing have led to the generation of NHP models for human diseases. Since 2014, there have been several reports of the generation of monkey models using transcription activator-like endonucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9); some of these NHP models showed symptoms that were much closer to those of human diseases than have been seen previously in mouse models. No off-targeting was observed in the NHP models, and multiple gene knockout and biallelic mutants were feasible with low efficiency. These findings suggest that there are many possibilities to establish NHP models for human diseases that can mimic human diseases more faithfully than rodent models.
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Targeted Gene Disruption in Pacific Oyster Based on CRISPR/Cas9 Ribonucleoprotein Complexes.
The Pacific oyster (Crassostrea gigas) is a representative bivalve mollusc that is widely cultured in the world. In recent years, it has become an important model species for ecological, evolutionary, and developmental studies because of its ability to survive in extreme environmental conditions as a sessile filter feeder and its classical mosaic pattern of development. Although the complete genome sequence of C. gigas is available and omics data have been rapidly generated for the past few years, the genetic tools for gene functional studies have thus far been limited to RNA interference technology. In this study, we developed a gene editing system for C. gigas based on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 ribonucleoprotein complexes. Two candidate genes, myostatin (MSTN) and Twist, were selected as targets. After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, CRISPR-induced indel mutations were detected in the target genes. The CRISPR/Cas9-induced mutations were predominantly small indel mutations ranging in size from 1 to 24 bp in these two target genes. These results demonstrate that CRISPR/Cas9 can be successfully used as an effective targeted gene editing system in C. gigas. The method reported here provides a powerful tool for gene functional studies in oysters and other marine bivalves, and potentially as a new technology for genetic engineering to improve oyster traits for aquaculture.
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Public health applications of CRISPR: How children's health can benefit.
Children under the age of five years old face significant mortality risks around the world. Public health innovations, particularly gene-editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) could help to reduce the risk of death in children under the age of five years old. For example, CRISPR-based strategies could reduce infectious disease morbidity by gene editing mosquitoes to prevent transmission of malaria. CRISPR gene editing technology could also help to screen for influenza virus and prevent it from replicating; influenza is a particularly difficult to treat and severe virus causing many deaths in children. The lack of liver, kidney, and heart donations for children on the organ donation waiting list could also benefit from CRISPR. Gene editing of pigs to reduce rejection rates and associated risks of porcine endogenous retroviruses could allow for the utilization of pig organs for transplant. Here we review proposed applications of gene-editing technology in public health and discuss its potential to reduce child mortality and morbidity globally.
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