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Minimally Humanized Ezh2 Exon-18 Mouse Cell Lines Validate Preclinical CRISPR/Cas9 Approach to Treat Weaver Syndrome.
Weaver syndrome is a rare neurodevelopmental disorder that encompasses macrocephaly, tall stature, obesity, brain anomalies, intellectual disability, and increased susceptibility to cancer. This dominant monogenic disorder is caused by germline variants in enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a key epigenetic writer. Unfortunately, there are no effective treatments for Weaver syndrome. However, preclinical results support the potential for therapeutic gains, despite the prenatal onset. Thus, for the first time, we tested whether CRISPR/Cas9 gene-editing strategies may be able to "correct" a Weaver syndrome variant at the DNA level. We initiated these preclinical studies by humanizing the region surrounding the most-common recurring patient-variant location in mouse embryonic stem cells (ESCs). Humanization ensures that DNA-binding strategies will be directly translatable to human cells and patients. We then introduced into ESCs the humanized region, but now carrying the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys, and characterized the enzymatic properties of this missense variant. Our data showed a significant and dramatic reduction in EZH2-enzymatic activity, supporting previous cell-free studies of this variant as well as in vitro and in vivo mouse work by other teams. Intriguingly, this most-common variant does not create a complete loss-of-function, but rather is a hypomorphic allele. Together with prior reports describing hypomorphic effects of missense EZH2 variants, these results demonstrate that the etiology of Weaver syndrome does not require complete loss of EZH2 enzymatic activity. Toward therapy, we tested four CRISPR gene-editing strategies. We demonstrated that Streptococcus pyogenes Cas9 (SpCas9) showed the highest variant correction (70.5%), but unfortunately also the highest alteration of the nonvariant allele (21.1-26.2%), an important consideration for gene-editing treatment of a dominant syndrome. However, Staphylococcus aureus Cas9 (SaCas9) gave a variant correction (52.5%) that was not significantly different than SpCas9, and encouragingly the lowest alteration of the nonvariant allele (2.0%). Thus, the therapeutic strategy using the small SaCas9 enzyme, a size that allows flexibility in therapeutic delivery, was the most optimal for targeting the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys.
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Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.
The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.
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Gene Modulation with CRISPR-based Tools in Human iPSC-Cardiomyocytes.
Precise control of gene expression (knock-out, knock-in, knockdown or overexpression) is at the heart of functional genomics - an approach to dissect the contribution of a gene/protein to the system's function. The development of a human in vitro system that can be patient-specific, induced pluripotent stem cells, iPSC, and the ability to obtain various cell types of interest, have empowered human disease modeling and therapeutic development. Scalable tools have been deployed for gene modulation in these cells and derivatives, including pharmacological means, DNA-based RNA interference and standard RNA interference (shRNA/siRNA). The CRISPR/Cas9 gene editing system, borrowed from bacteria and adopted for use in mammalian cells a decade ago, offers cell-specific genetic targeting and versatility. Outside genome editing, more subtle, time-resolved gene modulation is possible by using a catalytically "dead" Cas9 enzyme linked to an effector of gene transcription in combination with a guide RNA. The CRISPRi / CRISPRa (interference/activation) system evolved over the last decade as a scalable technology for performing functional genomics with libraries of gRNAs. Here, we review key developments of these approaches and their deployment in cardiovascular research. We discuss specific use with iPSC-cardiomyocytes and the challenges in further translation of these techniques.
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Precision Genome Editing in Domestic Animals: A New Era for Agriculture and Medicine.
Precision genome editing technologies have transformed the generation and application of genetically engineered livestock, positioning domestic animals as powerful models for agriculture, biomedicine, and basic research. Historically, genetic engineering in livestock was constrained by the absence of embryonic stem cells, low efficiency of homologous recombination, long gestation periods, and extended generation intervals. The advent of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR-Cas systems, has overcome many of these barriers by enabling efficient targeted genome modification. This review focuses on the application of genome editing technologies in mammalian livestock species, with pigs highlighted as a representative model. We discuss the historical development of livestock genetic engineering and summarise two main routes used to establish genome edited livestock models. Key technical considerations unique to domestic animals are also presented. Genome editing has accelerated the production of livestock models for human disease, xenotransplantation, reproductive biology, and agricultural improvement, including disease resistance, altered growth traits, and enhanced product composition. Emerging epigenome editing strategies further expand the toolkit by allowing locus-specific gene regulation without permanent DNA modification. Despite rapid technological progress, challenges remain, including mosaicism, off-target effects, limited ability to segregate unintended edits, and ethical and regulatory concerns surrounding food and biomedical applications. Continued refinement of genome editing strategies will be essential for maximizing the utility and responsible implementation of genome-edited livestock. Overall, precision genome editing represents a transformative platform with broad implications for sustainable agriculture, translational research, and global food security.
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Efficient cancer modeling through CRISPR-Cas9/HDR-based somatic precision gene editing in mice.
CRISPR-Cas9 has been used successfully to introduce indels in somatic cells of rodents; however, precise editing of single nucleotides has been hampered by limitations of flexibility and efficiency. Here, we report technological modifications to the CRISPR-Cas9 vector system that now allows homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic tissues to generate tumor models with high flexibility and efficiency. Somatic editing of either Kras or Pik3ca in both normal and hyperplastic mammary glands led to swift tumorigenesis. The resulting tumors shared some histological, transcriptome, and proteome features with tumors induced by lentivirus-mediated expression of the respective oncogenes, but they also exhibited some distinct characteristics, particularly showing less intertumor variation, thus potentially offering more consistent models for cancer studies and therapeutic development. Therefore, this technological advance fills a critical gap between the power of CRISPR technology and high-fidelity mouse models for studying human tumor evolution and preclinical drug testing.
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History, evolution and classification of CRISPR-Cas associated systems.
This chapter provides a detailed description of the history of CRISPR-Cas and its evolution into one of the most efficient genome-editing strategies. The chapter begins by providing information on early findings that were critical in deciphering the role of CRISPR-Cas associated systems in prokaryotes. It then describes how CRISPR-Cas had been evolved into an efficient genome-editing strategy. In the subsequent section, latest developments in the genome-editing approaches based on CRISPR-Cas are discussed. The chapter ends with the recent classification and possible evolution of CRISPR-Cas systems.
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Application of Genome Editing in Tomato Breeding: Mechanisms, Advances, and Prospects.
Plants regularly face the changing climatic conditions that cause biotic and abiotic stress responses. The abiotic stresses are the primary constraints affecting crop yield and nutritional quality in many crop plants. The advances in genome sequencing and high-throughput approaches have enabled the researchers to use genome editing tools for the functional characterization of many genes useful for crop improvement. The present review focuses on the genome editing tools for improving many traits such as disease resistance, abiotic stress tolerance, yield, quality, and nutritional aspects of tomato. Many candidate genes conferring tolerance to abiotic stresses such as heat, cold, drought, and salinity stress have been successfully manipulated by gene modification and editing techniques such as RNA interference, insertional mutagenesis, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9). In this regard, the genome editing tools such as CRISPR/Cas9, which is a fast and efficient technology that can be exploited to explore the genetic resources for the improvement of tomato and other crop plants in terms of stress tolerance and nutritional quality. The review presents examples of gene editing responsible for conferring both biotic and abiotic stresses in tomato simultaneously. The literature on using this powerful technology to improve fruit quality, yield, and nutritional aspects in tomato is highlighted. Finally, the prospects and challenges of genome editing, public and political acceptance in tomato are discussed.
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Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery.
Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.
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An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique.
Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.
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Genome Editing of Medaka.
Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knock-out strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knock-in using a plasmid carrying a sequence that does not cause off-target effects in medaka.
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