Frontiers in genome editing最新文献

Introduction: Copy number variation (CNV) is one of the crucial elements among genomic structural variations that span plant breeding. However, its impact on agricultural traits has remained elusive.

Methods: We modulated CNVs using two genome-editing technologies, CRISPR/Cas9 and Cas3, along with their verification methods in rice to elucidate the effect of CNVs and further harness to improve relevant agronomic traits.

Results: The addition of cytosine extension to the conventional single-guide RNA and its combination with Cas9 generated a frameshift mutation in parts of the OsGA20ox1 gene copies, substantially modifying its CNV. Phenotypes of the copy number variants revealed OsGA20ox1 copy number as a determinant of seedling vigor in rice. The Cas3 nuclease, which induces large-scale deletions, effectively decreased the copy number of the OsMTD1 gene. We verified the copy number of each gene by combining droplet digital polymerase chain reaction (ddPCR), Sanger sequencing, and bioinformatics tools.

Discussion: Altogether, the two technologies are expected to lay the foundation for new approaches to plant breeding by controlling CNV.

Genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with complex traits in poultry. However, most GWAS-identified variants reside in non-coding regions, making their functional relevance to their phenotypes unclear. Emerging evidence suggests that many of these markers overlap cis-regulatory elements, yet experimental validation of their biological function remains limited. Here, we investigated non-coding GWAS variants associated with nucleotide-related compounds in chicken breast muscle by targeting SNP-containing genomic regions using a CRISPR activation (CRISPRa) system in DF-1 cells and profiling transcriptomic responses via bulk RNA sequencing to assess the functional impact of activating these regions. Based on chicken muscle-specific epigenetic profiles and chromatin state annotations, we identified three significant GWAS variants on chromosome five associated with nucleotide metabolism. These variants are situated within cis-regulatory elements, specifically in intron three of DUSP8, intron one of SLC25A22, and upstream of FBXO3. To understand their functional impact, we employed an in vitro CRISPRa system with targeted guide RNAs to activate each non-coding SNP region in DF-1 cells. This activation resulted in significant changes at the transcriptomic level. Subsequent functional enrichment analysis of the differentially expressed genes consistently highlighted muscle-related pathways across all SNPs, including MAPK signaling, cytoskeletal remodeling, and ECM-receptor interactions, which are potentially involved in regulating nucleotide metabolism and deposition in muscle. Furthermore, transcript-level analysis of RNA-seq reads revealed that the non-coding SNP region within the intron three of DUSP8 may function as an alternative promoter, resulting in significantly higher expression of a shorter transcript that could generate a non-canonical protein isoform. Our study demonstrates that activating genomic regions harboring specific non-coding GWAS SNPs can modulate gene expression, suggesting that these SNPs may contribute to gene regulatory functions. Importantly, this work underscores the powerful utility of CRISPRa as a functional genomics tool for linking GWAS signals to their biological roles in chickens by targeting SNP-containing regions and uncovering consequential molecular phenotypes.

Tumor-associated antigen (TAA) loss remains a significant mechanism of resistance to chimeric antigen receptor (CAR) T cell therapy, leading to relapse in patients with B-cell malignancies and representing a major clinical challenge. Recent clinical data suggest that CD19 antigen loss triggers relapse in more than 40% of patients undergoing CD19 CAR-T cell therapy. To rigorously validate antigen loss, robust in vitro models that mimic the dynamic process of antigen escape are essential. However, the current absence of these models hampers our ability to fully evaluate and optimize treatment strategies. To model this clinically relevant phenomenon, we generated single (sKO), double (dKO), and triple (tKO) knockout Raji lymphoma cell lines targeting CD19, CD20, and CD22 using CRISPR/Cas9 genome editing. Initially, we established a dual-reporter cell line expressing the fluorescent marker mCherry and the bioluminescent marker Luciferase, enabling a uniform luminescence background across all the knockout cell lines before performing the CRISPR/Cas9 editing. The loss of individual or combinatorial TAAs was validated at the genomic, transcript, and protein levels. Functional co-culture assays with antigen-specific CAR-T cells showed that antigen-deficient Raji cells resisted CAR-T cell-mediated killing, closely mimicking clinical relapse. The triple knockout (tKO) model, in particular, provided a superior system compared to commonly used K562 models, as it retains the same lymphoma background while eliminating the crucial antigenic targets, thus better simulating resistance to CAR-T cell therapy. These antigen-loss models serve as valuable tools for studying mechanisms of CAR-T cell resistance and evaluating next-generation, multi-targeting CAR-T cell therapies.

Japan has rapidly deregulated certain types of agricultural genome editing, yet the societal integration of these products warrants further investigation. This paper analyzed the sale and people's perception of genome-edited food crops in Japan after reviewing the regulatory framework. Of four genome-edited crops approved as non-genetically modified organism, only one is sold online to consumers who credit safety information and perceive usefulness. Some consumers express deep safety concern, advocating mandatory labeling. The majority of people are not sufficiently aware of genome editing. To enhance informed consumer choices of genome-edited food crops, it is crucial to share visions in society, hold risk communication for mutual understanding, and maintain clear labels, including organic food standards.

In recent years gene therapy has emerged as a powerful technology for treatment of a large variety of inherited disorders. With the FDA approval of in vivo gene therapy of hemophilia A and B using AAV-mediated transgene delivery to hepatocytes, the path towards a new treatment era seemed paved. Also, CRISPR-Cas based approaches have reached the clinic, as in the ex vivo treatment of hematopoietic stem cells for sickle cell disease and thalassemia patients. The question arises whether these innovative strategies will also be suitable for patients with von Willebrand Disease (VWD). Whilst in and ex vivo delivery to endothelial cells (ECs) has been demonstrated, and CRISPR-Cas9 gene editing has been successful in ECs, there are currently no gene therapy options available for VWD. The wide variety of pathogenic VWF mutations makes development of broadly applicable, cost-effective gene therapies challenging. While delivery of von Willebrand factor (VWF) as a therapeutic transgene would be optimal, the size of VWF challenges efficient delivery. Therefore, treatment of VWD requires targeted, personalized gene therapy; for instance by using the newest CRISPR-Cas technologies which can be tailored to facilitate alteration and restoration of various pathogenic VWD variants. This review describes the inherited bleeding disorder VWD and potential gene therapy approaches for management of the disease. Thereby we are exploring different CRISPR-Cas technologies and recent developments in the field. Moreover, we will discuss the ongoing advances of in vivo delivery systems, all with the scope on ECs.

Homology-directed repair (HDR) holds great promise for plant genetic engineering but remains challenging due to its inherently low efficiency in gene editing applications. While studies in animal systems suggest that the structure of the donor repair template (DRT) influences HDR efficiency, this parameter remains largely unexplored in plants. In this study, we combined protoplast transfection with next-generation sequencing to analyse the impact of DRT structure on HDR efficiency in potato. A highly efficient ribonucleoprotein (RNP) complex targeting the soluble starch synthase 1 (SS1) gene was used in combination with various DRTs, differing in structural factors such as homology arm (HA) length, strandedness (i.e., ssDNA vs. dsDNA), and sequence orientation in ssDNA donors. Our results indicate that a ssDNA donor in the target orientation outperformed other configurations, achieving a HDR efficiency of 1.12% of the sequencing reads in the pool of protoplasts. Interestingly, HDR efficiency appeared independent of HA length. Notably, a ssDNA donor with HAs as short as 30 nucleotides led to targeted insertions in up to 24.89% of reads on average, but predominantly via alternative imprecise repair pathways, such as microhomology-mediated end joining (MMEJ). This donor structure also consistently yielded the highest HDR and targeted insertion rates at two out of three additional loci tested, offering valuable insights for future genome editing strategies in potato. We further assessed strategies to favour HDR over alternative repair outcomes, including the use of small molecules known to inhibit competing pathways in animal systems, and modifications to DRTs to enhance their availability in the vicinity of the target site. However, these approaches did not improve HDR efficiency. Overall, this study presents an effective platform for rapidly assessing gene editing components in potato and provides insights for achieving high-frequency, targeted insertions of short DNA fragments, especially relevant for efficient knock-in integration in non-coding genomic regions.

CRISPR-based technologies have revolutionized plant science by enabling precise modulation of gene function, including CRISPR activation (CRISPRa), a recently emerging strategy which shows particular promise for enhancing disease resistance through targeted gene upregulation. Unlike conventional CRISPR editing, which introduces double-stranded DNA breaks and permanent genomic changes, CRISPRa employs a deactivated Cas9 (dCas9) fused to transcriptional activators. This system allows quantitative and reversible gene activation without altering the DNA sequence, offering a gain-of-function (GOF) like enhanced blight resistance in staple crops. Despite its potential, the limited adoption of CRISPRa in plant biology to date underscores the need for future studies to fully harness its capabilities for crop improvement. This review addresses the groundbreaking and relatively underexplored potential of CRISPR activation (CRISPRa) systems for GOF studies in plant biology, and advocates for the adoption of CRISPRa to discover and harness genetic variation for enhancing disease resistance. We present recent advancements in CRISPRa technology, emphasizing its successful application in boosting plant immunity. Moreover, we discuss the synergistic potential of integrating CRISPRa with functional genomics tools such as genome-wide association studies (GWAS) and multi-omics approaches to identify and characterize key resistance genes. Additionally, we highlight ongoing progress in developing plant-specific programmable transcriptional activators (PTAs) to optimize CRISPRa efficiency. Challenges associated with achieving transgene-free overexpression and the deployment of alternative CRISPR systems are also explored. Together, these advances position CRISPRa as a transformative tool for future crop breeding strategies aimed at achieving durable, broad-spectrum disease resistance and sustainability in agriculture.