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Therapeutic genome editing with engineered nucleases.
Targeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.
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Addressing the dark matter of gene therapy: technical and ethical barriers to clinical application.
Gene therapies for genetic diseases have been sought for decades, and the relatively recent development of the CRISPR/Cas9 gene-editing system has encouraged a new wave of interest in the field. There have nonetheless been significant setbacks to gene therapy, including unintended biological consequences, ethical scandals, and death. The major focus of research has been on technological problems such as delivery, potential immune responses, and both on and off-target effects in an effort to avoid negative clinical outcomes. While the field has concentrated on how we can better achieve gene therapies and gene editing techniques, there has been less focus on when and why we should use such technology. Here we combine discussion of both the technical and ethical barriers to the widespread clinical application of gene therapy and gene editing, providing a resource for gene therapy experts and novices alike. We discuss ethical problems and solutions, using cystic fibrosis and beta-thalassemia as case studies where gene therapy might be suitable, and provide examples of situations where human germline gene editing may be ethically permissible. Using such examples, we propose criteria to guide researchers and clinicians in deciding whether or not to pursue gene therapy as a treatment. Finally, we summarize how current progress in the field adheres to principles of biomedical ethics and highlight how this approach might fall short of ethical rigour using examples in the bioethics literature. Ultimately by addressing both the technical and ethical aspects of gene therapy and editing, new frameworks can be developed for the fair application of these potentially life-saving treatments.
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Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.
Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.
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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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Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells.
The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.
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An Electrochemical Protocol for CRISPR-Mediated Gene-Editing of Sheep Embryonic Fibroblast Cells.
Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.
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Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars.
CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.
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Application of CRISPR/Cas9-Based Genome Editing in Studying the Mechanism of Pandrug Resistance in Klebsiella pneumoniae.
In this study, a CRISPR/Cas9-mediated genome editing method was used to study the functions of the mgrB, tetA, and ramR genes in mediating colistin and tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP). Inactivation of the tetA or ramR gene or the mgrB gene by CRISPR/Cas9 affected bacterial susceptibility to tigecycline or colistin, respectively. This study proved that the CRISPR/Cas9-based genome editing method could be effectively applied to K. pneumoniae and should be further utilized for genetic characterization.
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DeepCRISTL: deep transfer learning to predict CRISPR/Cas9 functional and endogenous on-target editing efficiency.
MOTIVATION: CRISPR/Cas9 technology has been revolutionizing the field of gene editing in recent years. Guide RNAs (gRNAs) enable Cas9 proteins to target specific genomic loci for editing. However, editing efficiency varies between gRNAs. Thus, computational methods were developed to predict editing efficiency for any gRNA of interest. High-throughput datasets of Cas9 editing efficiencies were produced to train machine-learning models to predict editing efficiency. However, these high-throughput datasets have low correlation with functional and endogenous editing. Another difficulty arises from the fact that functional and endogenous editing efficiency is more difficult to measure, and as a result, functional and endogenous datasets are too small to train accurate machine-learning models on. RESULTS: We developed DeepCRISTL, a deep-learning model to predict the on-target efficiency given a gRNA sequence. DeepCRISTL takes advantage of high-throughput datasets to learn general patterns of gRNA on-target editing efficiency, and then uses transfer learning (TL) to fine-tune the model and fit it to the functional and endogenous prediction task. We pre-trained the DeepCRISTL model on more than 150 000 gRNAs, produced through the DeepHF study as a high-throughput dataset of three Cas9 enzymes. We improved the DeepHF model by multi-task and ensemble techniques and achieved state-of-the-art results over each of the three enzymes: up to 0.89 in Spearman correlation between predicted and measured on-target efficiencies. To fine-tune model weights to predict on-target efficiency of functional or endogenous datasets, we tested several TL approaches, with gradual learning being the overall best performer, both when pre-trained on DeepHF and when pre-trained on CRISPROn, another high-throughput dataset. DeepCRISTL outperformed state-of-the-art methods on all functional and endogenous datasets. Using saliency maps, we identified and compared the important features learned by the model in each dataset. We believe DeepCRISTL will improve prediction performance in many other CRISPR/Cas9 editing contexts by leveraging TL to utilize both high-throughput datasets, and smaller and more biologically relevant datasets, such as functional and endogenous datasets. AVAILABILITY AND IMPLEMENTATION: DeepCRISTL is available via github.com/OrensteinLab/DeepCRISTL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Evaluation of Bna.SCT and Bna.REF1 as Target Genes to Reduce Sinapine in Rapeseed Using a Protoplast-Based CRISPR RNP Approach.
Rapeseed is a major oil crop worldwide, producing both oil and a high amount of protein. However, the use of its seed meal as a protein source for animal feed is limited by antinutritional factors, such as sinapine, which reduces nutrient absorption and affects the palatability. Efforts to reduce sinapine levels through conventional breeding have had limited success. Given the challenges of a changing climate and a growing global population, maximising crop utility, particularly the value of seed meal as a byproduct, is increasingly important. Genetic modification has been successfully used to reduce sinapine in rapeseed, but regulatory restrictions limit its commercial adoption in some regions. CRISPR-Cas gene editing, which is gaining broader global acceptance, offers a promising alternative to directly produce transgene-free mutants. In this study, we build on our previous work by generating transgene-free rapeseed mutants using protoplast-based CRISPR RNP gene editing. We successfully targeted the Bna.SCT and Bna.REF1 genes with editing efficiencies of 22%-63%, frequently achieving mutations in all four alleles of the target genes in T2 plants with a single sgRNA. Seed sinapine content was reduced by up to 38% in Bna.SCT mutants and 77% in Bna.REF1 mutants, with no observed effects on plant growth or development. These findings suggest that Bna.REF1 is the most effective target for sinapine reduction in transgene-free mutants among the genes tested in our lab.
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