Frontiers in genome editing最新文献

NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

Considerable effort has been devoted to developing adeno-associated virus (AAV)-based vectors for gene therapy in cystic fibrosis (CF). As a result of directed evolution and capsid shuffling technology, AAV capsids are available with widespread tropism for airway epithelial cells. For example, AAV2.5T and AAV6.2 are two evolved capsids with improved airway epithelial cell transduction properties over their parental serotypes. However, limited research has been focused on identifying their specific cellular tropism. Restoring cystic fibrosis transmembrane conductance regulator (CFTR) expression in surface columnar epithelial cells is necessary for the correction of the CF airway phenotype. Basal cells are a progenitor population of the conducting airways responsible for replenishing surface epithelial cells (including secretory cells and ionocytes), making correction of this cell population vital for a long-lived gene therapy strategy. In this study, we investigate the tropism of AAV capsids for three cell types in primary cultures of well-differentiated human airway epithelial (HAE) cells and primary human airway basal cells. We observed that AAV2.5T transduced surface epithelial cells better than AAV6.2, while AAV6.2 transduced airway basal cells better than AAV2.5T. We also investigated a recently developed capsid, AAV6.2FF, which has two surface tyrosines converted to phenylalanines. Next, we incorporated reciprocal mutations to create AAV capsids with further improved surface and basal cell transduction characteristics. Lastly, we successfully employed a split-intein approach using AAV to deliver an adenine base editor (ABE) to repair the CFTR ^R553X mutation. Our results suggest that rational incorporation of AAV capsid mutations improves AAV transduction of the airway surface and progenitor cells and may ultimately lead to improved pulmonary function in people with CF.

Parasitic plants pose a significant threat to global agriculture, causing substantial crop losses and hampering food security. In recent years, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology has emerged as a promising tool for developing resistance against various plant pathogens. Its application in combating parasitic plants, however, remains largely unexplored. This review aims to summarise current knowledge and research gaps in utilising CRISPR to develop resistance against parasitic plants. First, we outline recent improvements in CRISPR gene editing tools, and what has been used to combat various plant pathogens. To realise the immense potential of CRISPR, a greater understanding of the genetic basis underlying parasitic plant-host interactions is critical to identify suitable target genes for modification. Therefore, we discuss the intricate interactions between parasitic plants and their hosts, highlighting essential genes and molecular mechanisms involved in defence response and multilayer resistance. These include host resistance responses directly repressing parasitic plant germination or growth and indirectly influencing parasitic plant development via manipulating environmental factors. Finally, we evaluate CRISPR-mediated effectiveness and long-term implications for host resistance and crop improvement, including inducible resistance response and tissue-specific activity. In conclusion, this review highlights the challenges and opportunities CRISPR technology provides to combat parasitic plants and provides insights for future research directions to safeguard global agricultural productivity.

DNA regulatory elements intricately control when, where, and how genes are activated. Therefore, understanding the function of these elements could unveil the complexity of the genetic regulation network. Genome-wide significant variants are predominantly found in non-coding regions of DNA, so comprehending the predicted functional regulatory elements is crucial for understanding the biological context of these genomic markers, which can be incorporated into breeding programs. The emergence of CRISPR technology has provided a powerful tool for studying non-coding regulatory elements in genomes. In this study, we leveraged epigenetic data from the Functional Annotation of Animal Genomes project to identify promoter and putative enhancer regions associated with three genes (HBBA, IRF7, and PPARG) in the chicken genome. To identify the enhancer regions, we designed guide RNAs targeting the promoter and candidate enhancer regions and utilized CRISPR activation (CRISPRa) with dCas9-p300 and dCas9-VPR as transcriptional activators in chicken DF-1 cells. By comparing the expression levels of target genes between the promoter activation and the co-activation of the promoter and putative enhancers, we were able to identify functional enhancers that exhibited augmented upregulation. In conclusion, our findings demonstrate the remarkable efficiency of CRISPRa in precisely manipulating the expression of endogenous genes by targeting regulatory elements in the chicken genome, highlighting its potential for functional validation of non-coding regions.

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal settings, such as teaching labs. Gene-specific targeting sequences can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic or transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) genes, we were able to create a collection of predicted null mutations in all nine family members with little effort. We then explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. In our hands, multiplexing was associated with pronounced disparities: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species, thereby preventing a systematic mutagenesis of the family.

Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T₀ plants. Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T₀ plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.

The first genetically modified large animals were developed in 1985 by microinjection to increase the growth of agricultural livestock such as pigs. Since then, it has been a difficult trail due to the lack of genetic tools. Although methods and technologies were developed quickly for the main experimental mammal, the mouse, e.g., efficient pronuclear microinjection, gene targeting in embryonic stem cells, and omics data, most of it was-and in part still is-lacking when it comes to livestock. Over the next few decades, progress in genetic engineering of large animals was driven less by research for agriculture but more for biomedical applications, such as the production of pharmaceutical proteins in the milk of sheep, goats, or cows, xeno-organ transplantation, and modeling human diseases. Available technologies determined if a desired animal model could be realized, and efficiencies were generally low. Presented here is a short review of how genome editing tools, specifically CRISPR/Cas, have impacted the large animal field in recent years. Although there will be a focus on genome engineering of pigs for biomedical applications, the general principles and experimental approaches also apply to other livestock species or applications.

Plant genome editing, a recently discovered method for targeted mutagenesis, has emerged as a promising tool for crop improvement and gene function research. Many genome-edited plants, such as rice, wheat, and tomato, have emerged over the last decade. As the preliminary steps in the procedure for genome editing involve genetic transformation, amenability to genome editing depends on the efficiency of genetic engineering. Hence, there are numerous reports on the aforementioned crops because they are transformed with relative ease. Legume crops are rich in protein and, thus, are a favored source of plant proteins for the human diet in most countries. However, legume cultivation often succumbs to various biotic/abiotic threats, thereby leading to high yield loss. Furthermore, certain legumes like peanuts possess allergens, and these need to be eliminated as these deprive many people from gaining the benefits of such crops. Further genetic variations are limited in certain legumes. Genome editing has the potential to offer solutions to not only combat biotic/abiotic stress but also generate desirable knock-outs and genetic variants. However, excluding soybean, alfalfa, and Lotus japonicus, reports obtained on genome editing of other legume crops are less. This is because, excluding the aforementioned three legume crops, the transformation efficiency of most legumes is found to be very low. Obtaining a higher number of genome-edited events is desirable as it offers the option to genotypically/phenotypically select the best candidate, without the baggage of off-target mutations. Eliminating the barriers to genetic engineering would directly help in increasing genome-editing rates. Thus, this review aims to compare various legumes for their transformation, editing, and regeneration efficiencies and discusses various solutions available for increasing transformation and genome-editing rates in legumes.

Inactivation or targeted disruption of a gene provides clues to assess the function of the gene in many cellular processes. Knockdown or knocking out a gene has been widely used for this purpose. However, recently CRISPR mediated genome editing has taken over the knockout/knockdown system with more precision. CRISPR technique has enabled us to perform targeted mutagenesis or genome editing to address questions in fundamental biology to biomedical research. Its application is wide in understanding the role of genes in the disease process, and response to therapy in cancer, metabolic disorders, or infectious disease. In this article, we have focused on infectious disease and how genome-wide CRISPR screens have enabled us to identify host factors involved in the process of infection. Understanding the biology of the host-pathogen interaction is of immense importance in planning host-directed therapy to improve better management of the disease. Genome-wide CRISPR screens provide strong mechanistic ways to identify the host dependency factors involved in various infections. We presented insights into genome-wide CRISPR screens conducted in the context of infectious diseases both viral and bacterial that led to better understanding of host-pathogen interactions and immune networks. We have discussed the advancement of knowledge pertaining to influenza virus, different hepatitis viruses, HIV, most recent SARS CoV2 and few more. Among bacterial diseases, we have focused on infection with life threatening Mycobacteria, Salmonella, S. aureus, etc. It appears that the CRISPR technique can be applied universally to multiple infectious disease models to unravel the role of known or novel host factors.