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Applications of CRISPR-Cas-Based Genome Editing Approaches Against Human Cytomegalovirus Infection.
Human cytomegalovirus (HCMV), a globally ubiquitous herpesvirus with the ability to carry out both lytic productive and lifelong latent infections, is a major cause of congenital infections, often leading to intellectual disabilities and neurological disorders. Moreover, HCMV is an opportunistic pathogen commonly found in immunocompromised individuals such as organ transplant recipients, HIV-positive individuals, and cancer patients, causing severe and life-threatening complications. While effective in inhibiting viral lytic infection, current FDA-approved compounds cannot eliminate the latent viral genome and have little effect on viral latent infection. Developing novel antiviral therapeutic approaches to eliminate HCMV lytic and latent infections is a major public health priority for controlling HCMV infection and preventing viral-associated diseases. The genome-editing technology based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) RNA-guided nuclease system represents a novel and promising antiviral approach through modifying or destroying the genetic material of human viruses. This review summarizes the recently published progress in using the CRISPR-Cas approach to study and inhibit HCMV infections and discusses prospects for developing the CRISPR-based genome-editing technology for therapeutic applications against HCMV infection and associated diseases.
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Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus.
CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.
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TKC-MC: An Effective Strategy for Generating Heritable Heterozygous Mutations in Essential Genes in Rice.
The CRISPR/Cas9 gene-editing technology has been widely used in defining gene functions and crop improvement. However, some genes are essential for plant growth and development. Loss-of-function homozygous mutations in essential genes lead to plant death or sterility. Mutations in essential genes need to be maintained and propagated in heterozygous plants. CRISPR/Cas9 technology is highly efficient in generating homozygous or bi-allelic mutations at T0 generation in rice, making it difficult to generate useful genetic materials for essential genes using traditional gene editing technology. In this study, we designed Transgene-Killer CRISPR (TKC)-mediated mismatch-spacer targeting (TKC-M) to efficiently generate heritable heterozygous mutations in essential genes in rice. Leveraging our earlier transgenic offspring self-elimination TKC platform, TKC-M relied on timely self-elimination of Cas9 and engineered gRNA-target mismatches to enrich heritable heterozygous or mosaic incomplete-edited T0 mutants and heterozygous progeny. We found that the sensitivity of targets to spacer mismatch(es) varies. A single-base mismatch at gRNA positions 11 or 17 yielded abundant heritable heterozygotes in sensitive targets. For insensitive targets, dual mismatches at positions 8 and 15 maximised heritable heterozygotes. Co-transformation of rice with TKC vectors carrying gRNA without mismatches (G1), gRNA with a mismatch at position 11 (M11) and M8 + M15 spacers, termed TKC-M Cocktail (TKC-MC) significantly increased the incomplete-edited mutant ratio compared with using G1 alone. This work establishes a technical foundation for generating mutant libraries that cover every single gene in a plant genome and for in-depth research on essential genes.
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Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA.
CRISPR editors including nucleases, base editors, and prime editors can efficiently correct disease-causing genetic variants or disrupt target genes. Editing outcomes are commonly evaluated in cultured primary cells, patient-derived cells, or engineered cell lines to study the impact of genetic variation or as a first step before initiating animal studies or clinical translation. Delivery of editors as mRNA together with synthetic guide RNAs into mammalian cells can improve editing efficiency relative to plasmid-based approaches and prevent issues such as DNA integration or off-target editing from sustained expression. This article presents a workflow to prepare genome editor mRNA by in vitro transcription (IVT), including co-transcriptional capping and chemically-modified nucleotides, electroporate editor mRNA and guide RNAs into primary human fibroblasts, induced pluripotent stem cells (iPSCs), or lymphoblastoid cell lines (LCLs), and quantify editing outcomes by targeted amplicon sequencing on an Illumina platform followed by analysis using CRISPResso2. This workflow enables quantitative benchmarking of guide RNAs, electroporation parameters, and editor variants, and supports downstream applications including single-cell cloning, phenotypic assays, preclinical animal studies, and therapeutic development.
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SunTag-PE: a modular prime editing system enables versatile and efficient genome editing.
Prime editing (PE) holds tremendous potential in the treatment of genetic diseases because it can install any desired base substitution or local insertion/deletion. However, the full-length PE effector size (6.3-kb) is beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical translation. Various splitting strategies have been used to improve its delivery, but always accompanied by compromised PE efficiency. Here, we developed a modular and efficient SunTag-PE system that splits PE effectors into GCN4-nCas9 and single-chain variable fragment (scFv) tethered reverse transcriptase (RT). We observed that SunTag-PEs with 1×GCN4 in the N terminus of nCas9 was the most efficient configuration rather than multiple copies of GCN4. This SunTag-PE strategy achieved editing levels comparable to canonical fused-PE (nCas9 and RT are linked together) and higher than other split-PE strategies (including sPE and MS2-PE) in both PE2 and PE3 forms with no increase in insertion and deletion (indel) byproducts. Moreover, we successfully validated the modularity of SunTag-PE system in the Cas9 orthologs of SauCas9 and FrCas9. Finally, we employed dual AAVs to deliver SunTag-ePE3 and efficiently corrected the pathogenic mutation in HBB mutant cell line. Collectively, our SunTag-PE system provides an efficient modular splitting strategy for prime editing and further facilitate its transformation in clinics.
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Efficient A·T to G·C base conversions in dicots using adenine base editors expressed under the tomato EF1α promoter.
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m6A Editing: New Tool to Improve Crop Quality?
N6-methyladenosine (m6A) is the most common type of eukaryotic mRNA modification. It plays an important role in regulating plant growth and development and stress resistance. m6A modification influences nearly all aspects of RNA metabolism and functionality and has great potential for improving crop quality. However, changing m6A modification levels as a whole may have unpredictable effects, making it impossible to accurately predict the effect of specific m6A modifications on RNA. In this opinion article, the main challenges and possible solutions for exploring m6A modification functions in plant systems are discussed. An m6A editing platform that uses new high-throughput methods to identify m6A modification at single-base resolution, and genome editing for selective editing of specific m6A sites for crop improvement is proposed.
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CRISPR/Cas9-based editing of NF-YC4 promoters yields high-protein rice and soybean.
Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts. Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs. Building upon our prior research demonstrating the protein-boosting effects of the transcription factor NF-YC4, we identified conserved motifs targeted by RAV and WRKY repressors in the NF-YC4 promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increased NF-YC4 expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein. Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality.
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Nuclear receptor corepressors Ncor1 and Ncor2 (Smrt) are required for retinoic acid-dependent repression of Fgf8 during somitogenesis.
Retinoic acid (RA) repression of Fgf8 is required for many different aspects of organogenesis, however relatively little is known about how endogenous RA controls gene repression as opposed to gene activation. Here, we show that nuclear receptor corepressors NCOR1 and NCOR2 (SMRT) redundantly mediate the ability of RA to repress Fgf8. Ncor1;Ncor2 double mutants generated by CRISPR/Cas9 gene editing exhibited a small somite and distended heart phenotype similar to that of RA-deficient Raldh2-/- embryos, associated with increased Fgf8 expression and FGF signaling in caudal progenitors and heart progenitors. Embryo chromatin immunoprecipitation studies revealed that NCOR1/2 but not coactivators are recruited to the Fgf8 RA response element (RARE) in an RA-dependent manner, whereas coactivators but not NCOR1/2 are recruited RA-dependently to a RARE near Rarb that is activated by RA. CRISPR/Cas9-mediated genomic deletion of the Fgf8 RARE in mouse embryos often resulted in a small somite defect with Fgf8 derepression caudally, but no defect was observed in heart development or heart Fgf8 expression. This suggests the existence of another DNA element whose function overlaps with the Fgf8 RARE to mediate Fgf8 repression by RA and NCOR1/2. Our studies support a model in which NCOR1/2 mediates direct RA-dependent repression of Fgf8 in caudal progenitors in order to control somitogenesis.
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Genome editing retraces the evolution of toxin resistance in the monarch butterfly.
Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness1. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits2-4, and new genome editing technology is facilitating functional validation of these mutations in whole organisms1,5. We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins6-11. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na+/K+-ATPase)7-11, the physiological target of cardiac glycosides12. Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization13,14. We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage11,15. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides16, culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators17-19. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis13,14,20-22. Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities2,6-11,15,17-19.
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