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Generating CRISPR Alleles in Drosophila.
CRISPR gene editing is a versatile and efficient approach for generating a wide variety of genetic reagents in flies. This unparalleled ability to manipulate the genome has revolutionized neuroscience, allowing Drosophila neurobiologists to readily generate new alleles to probe gene function, investigate the functional consequences of disease-associated variants, tag endogenous proteins to follow their dynamic localization in neurons and glia, and much more. Here, we provide a comprehensive protocol for generating heritable mutations in Drosophila We particularly focus on design considerations and tips for avoiding common errors to maximize the likelihood of successful gene editing.
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Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".
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A Survey of Validation Strategies for CRISPR-Cas9 Editing.
The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones.
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Yesterday's Child: How Gene Editing for Enhancement Will Produce Obsolescence-and Why It Matters.
Despite the advent of CRISPR, safe and effective gene editing for human enhancement remains well beyond our current technological capabilities. For the discussion about enhancing human beings to be worth having, then, we must assume that gene-editing technology will improve rapidly. However, rapid progress in the development and application of any technology comes at a price: obsolescence. If the genetic enhancements we can provide children get better and better each year, then the enhancements granted to children born in any given year will rapidly go out of date. Sooner or later, every modified child will find him- or herself to be "yesterday's child." The impacts of such obsolescence on our individual, social, and philosophical self-understanding constitute an underexplored set of considerations relevant to the ethics of genome editing.
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Analysis of microsatellite instability in CRISPR/Cas9 editing mice.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated (Cas) protein 9 system is a novel and powerful tool which is widely used for genome editing. CRISPR/Cas9 is RNA-guided and can lead to desired genomic modifications. However, whether the CRISPR/Cas9-mediated genome editing causes genomic alterations and genomic instability, such as microsatellite instability (MSI), is still unknown. Here we detected MSI in 21 CRISPR/Cas9 mouse strains using a panel of 42 microsatellite loci which were selected from our previous studies. Surprisingly, MSI occurrence was common in CRISPR/Cas9 modified genome, and most of the strains (19/21, 90.5%) examined showed MSI. Of 42 loci examined, 8 loci (8/42, 19.05%) exhibited MSI in the Cas9 editing mice. The Ttll9 (4/42, 9.5%) were the most unstable strains, and D10Mit3 and D10Mit198 (9/21, 42.9%) were considered to be the most "hot" loci in the Cas9 strains we tested. Through analyzing the mutation of microsatellite loci, we provide new insights into the genomic alterations of CRISPR/Cas9 models and it will help us for a better understanding of this powerful technology.
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[Perspectives of genome editing in otorhinolaryngology].
BACKGROUND: Recent advances in DNA sequencing technology have enabled researchers to identify the genetic background underlying human illness. In addition, the latest genome editing technology, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9), provides great potential to edit genomic DNA sequences precisely with high efficiency. This technology has been evaluated for treatment of genetic diseases in recently published preclinical studies. Since many such genetic disorders can affect functional structures in the head and neck area, the technology bears high therapeutic potential in otorhinolaryngology. OBJECTIVE: In this article, we summarize the concept of CRISPR-Cas9-based therapies, recent achievements in preclinical applications, and future challenges for the implementation of this technology in otolaryngology. MATERIALS AND METHODS: Genetic targeting strategies were analyzed or established using genome sequencing data derived from online databases and literature. RESULTS: Recent research on animal models has shown that genome editing can be used to treat genetic diseases by specifically targeting mutant genomic loci. For example, one preclinical study in the field of otolaryngology has demonstrated that inherited autosomal dominant deafness in mice can be treated using CRISPR-Cas9. Moreover, the same strategies can be used to establish applications for the treatment of head and neck cancer. The greatest challenge appears to be establishment of a system for the safe and efficient delivery of therapeutic nucleotides in clinics. CONCLUSIONS: In theory, genome editing could be used in otolaryngology to target disease-causing genomic loci specifically. However, various challenges have to be overcome until applications can be used clinically.
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Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos.
Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)," has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.
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Black Swans of CRISPR: Stochasticity and Complexity of Genetic Regulation.
Recent waves of controversies surrounding genetic engineering have spilled into popular science in Twitter battles between reputable scientists and their followers. Here, a cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies is presented, focusing in particular on the stochastic nature of cellular control processes.
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Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.
CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.
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Maize mutant screens: from classical methods to new CRISPR-based approaches.
Mutations play a pivotal role in shaping the trajectory and outcomes of a species evolution and domestication. Maize (Zea mays) has been a major staple crop and model for genetic research for more than 100 yr. With the arrival of site-directed mutagenesis and genome editing (GE) driven by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), maize mutational research is once again in the spotlight. If we combine the powerful physiological and genetic characteristics of maize with the already available and ever increasing toolbox of CRISPR-Cas, prospects for its future trait engineering are very promising. This review aimed to give an overview of the progression and learnings of maize screening studies analyzing forward genetics, natural variation and reverse genetics to focus on recent GE approaches. We will highlight how each strategy and resource has contributed to our understanding of maize natural and induced trait variability and how this information could be used to design the next generation of mutational screenings.
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