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Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects.
BACKGROUND: CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies, revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design. RESULTS: We systematically compare specific targeting and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNAs cause pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerges as the most efficient tool for stable promoter targeting, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations. CONCLUSIONS: Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.
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An RNA-Directed Gene Editing Strategy for Attenuating the Infectious Potential of Feline Immunodeficiency Virus-Infected Cells: A Proof of Concept.
Modern antiretroviral therapy for immunodeficiency viruses, although remarkably effective in controlling viral transcription, and overt virus-associated morbidity, has failed to absolutely eradicate retroviruses from their infected hosts as a result of proviral integration in long-lived reservoir cells. Immunodeficiency virus-infected patients are therefore consigned to lifelong antiviral therapy as a means to control viremia, viral transmission, and infection-associated morbidity. Unfortunately, lifelong antiviral therapies can be difficult for patients to continuously maintain and may be associated with therapy-specific morbidities. Patient advocates have argued for new methods to achieve retroviral eradication. As a proof-of-concept study, a lentivirus-delivered RNA-directed gene editing strategy was utilized in a series of in vitro experiments in an attempt to attenuate the feline immunodeficiency virus (FIV) proviral load, viral transcription, and production of infectious virions. We found that a feline T lymphocyte cell line (MCH5-4) treated with an FIV-specific clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing tool resulted in a reduction of cell-free viral RNA relative to control cells. Decreased infectious potential was demonstrated in a two-step FIV infection study-naïve MCH5-4 cells infected with cell-free FIV harvested from FIV-infected and CRISPR lentivirus-treated cells had less integrated proviral DNA than control cells. This study represents the initial steps towards the development of an effective method of proviral eradication in an immunodeficiency virus-infected host.
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Fine-tuning epigenome editors.
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Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.
Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.
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Direct preparation of Cas9 ribonucleoproteins with an extended 'gRNA-shRNA' construct in Escherichia coli for precise genome manipulation.
Gene perturbation approaches have emerged as powerful tools for elucidating gene function and treating hereditary disorders. Previously, we developed a method for streamlined production of ready-to-use Cas9 ribonucleoproteins (RNPs) in Escherichia coli BL21(DE3). In this study, we present an improved approach by assembling Cas9 RNPs with an extended 'gRNA-shRNA' construct in the RNase III deficient strain HT115(DE3). Transfection of these engineered Cas9 RNPs into mammalian cells enables multidimensional genome manipulation, including simultaneous knockdown and knockout of target genes. Furthermore, the design of shRNA specifically targeting human DNA ligase IV (LIG4) significantly enhances efficiency in homology-directed repair genome editing. Collectively, our findings establish a user-friendly CRISPR/Cas9 RNP tool with immense potential for precise genome editing, gene function analysis, and gene therapy.
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Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.
CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.
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Reboot ethics governance in China.
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Messenger RNA and guide RNA distributions in lipid nanoparticles impact gene-editing efficiency in vivo.
Lipid nanoparticles (LNPs) are among the most advanced non-viral vectors for CRISPR-based gene-editing therapeutics. Co-packaging of messenger RNA (mRNA) and guide RNA (gRNA) inherently produces heterogeneous payload distributions. The impact of this heterogeneity on editing performance remains unclear. Here, we utilize cylindrical illumination confocal spectroscopy (CICS) for single-particle interrogation of ALC-0315 and DLin-MC3-DMA LNPs prepared by three different mixing methods. CICS resolves four distinct subpopulations: co-encapsulated (50.7%-60.4%), gRNA only (30.0%-36.5%), mRNA only (2.0%-3.4%), and empty (4.2%-13.8%), and it uncovers broad, particle-to-particle variability in RNA copy number within each class. Structure-function analysis reveals that LNP formulation and mixing processes influence payload distribution, resulting in a negative correlation between the fraction of empty LNPs and RNA loading per particle. We further investigated the correlation between these quality attributes and therapeutic performance. In mice, ALC-0315 LNPs carrying higher cargo loads (9.8 vs. 8.0 mRNA copies and 25.4 vs. 20.3 gRNA copies per co-encapsulated particle) yielded 1.5-fold higher in vivo editing activity (55.4% vs. 36.3% insertions and deletions [indels]) despite nearly identical biophysical characteristics including LNP size and RNA encapsulation. These results establish payload distribution as a potential determinant of gene-editing potency and demonstrate single-particle CICS as a powerful tool for rational design of multi-component nucleic acid-delivery systems.
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An undergraduate research experience in CRISPR-Cas9 mediated eukaryotic genome editing to teach fundamental biochemistry techniques.
CRISPR-Cas9 technology is an established, powerful tool for genome editing through the ability to target specific DNA sequences of interest for introduction of desired genetic modifications. CRISPR-Cas9 is utilized for a variety of purposes, ranging from a research molecular biology tool to treatment for human diseases. Due to its prominence across a variety of applications, it is critical that undergraduates in the life sciences are educated on CRISPR-Cas9 technology. To this end, we created an intensive eight-week long course-based undergraduate research experience (CURE) designed for students to understand CRISPR-Cas9 genome editing and perform it in Saccharomyces cerevisiae. Students enrolled in the CURE participate in 2, 3-h sessions a week and are engaged in the entire process of CRISPR-Cas9 genome editing, from preparation of genome editing reagents to characterization of mutant yeast strains. During the process, students master fundamental techniques in the life sciences, including sterile technique, Polymerase Chain Reaction (PCR), primer design, sequencing requirements, and data analysis. The course is developed with flexibility in the schedule for repetition of techniques in the event of a failed experiment, providing an authentic research experience for the students. Additionally, we have developed the course to be easily modified for the editing of any yeast gene, offering the potential to expand the course in research-driven classroom or laboratory settings.
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Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing.
The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc.
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