Frontiers in Genome Editing

Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4 h per day for 3 days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.

Satoru Sukegawa

Osamu Nureki

Seiichi Toki

Hiroaki Saika

Front. Genome Ed.

Genome editing in rice mediated by miniature size Cas nuclease SpCas12f

水稻中微型尺寸Cas核酸酶SpCas12f介导的基因组编辑

The 21st century witnessed a boom in plant genomics and gene characterization studies through RNA interference and site-directed mutagenesis. Specifically, the last 15 years marked a rapid increase in discovering and implementing different genome editing techniques. Methods to deliver gene editing reagents have also attempted to keep pace with the discovery and implementation of gene editing tools in plants. As a result, various transient/stable, quick/lengthy, expensive (requiring specialized equipment)/inexpensive, and versatile/specific (species, developmental stage, or tissue) methods were developed. A brief account of these methods with emphasis on recent developments is provided in this review article. Additionally, the strengths and limitations of each method are listed to allow the reader to select the most appropriate method for their specific studies. Finally, a perspective for future developments and needs in this research area is presented.

Sachin Rustgi

Salman Naveed

Jonathan Windham

Huan Zhang

Gözde S. Demirer

Plant biomacromolecule delivery methods in the 21st century

21世纪植物生物大分子传递方法

The successful application of nanobiotechnology in biomedicine has greatly changed the traditional way of diagnosis and treating of disease, and is promising for revolutionizing the traditional plant nanobiotechnology. Over the past few years, nanobiotechnology has increasingly expanded into plant research area. Nanomaterials can be designed as vectors for targeted delivery and controlled release of fertilizers, pesticides, herbicides, nucleotides, proteins, etc. Interestingly, nanomaterials with unique physical and chemical properties can directly affect plant growth and development; improve plant resistance to disease and stress; design as sensors in plant biology; and even be used for plant genetic engineering. Similarly, there have been concerns about the potential biological toxicity of nanomaterials. Selecting appropriate characterization methods will help understand how nanomaterials interact with plants and promote advances in plant nanobiotechnology. However, there are relatively few reviews of tools for characterizing nanomaterials in plant nanobiotechnology. In this review, we present relevant imaging tools that have been used in plant nanobiotechnology to monitor nanomaterial migration, interaction with and internalization into plants at three-dimensional lengths. Including: 1) Migration of nanomaterial into plant organs 2) Penetration of nanomaterial into plant tissues (iii)Internalization of nanomaterials by plant cells and interactions with plant subcellular structures. We compare the advantages and disadvantages of current characterization tools and propose future optimal characterization methods for plant nanobiotechnology.

Bin Zhao

Zhongxu Luo

Honglu Zhang

Imaging tools for plant nanobiotechnology

植物纳米生物技术的成像工具

The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.

Kelsey M. Reed

Bastiaan O. R. Bargmann

Protoplast Regeneration and Its Use in New Plant Breeding Technologies

In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.

Jin-Jun Yue

Jin-Ling Yuan

Fu-Hui Wu

Yu-Hsuan Yuan

Qiao-Wei Cheng

Chen-Tran Hsu

Choun-Sea Lin

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

Sugarcane ( Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

Eleanor J. Brant

David May

Ayman Eid

Fredy Altpeter

Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane

高多倍体甘蔗中靶向CRISPR/Cas突变检测的基因分型分析比较

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.

Sarkar Sardar Azeez

Rahin Shareef Hamad

Bahra Kakamin Hamad

Mudhir Sabir Shekha

Peter Bergsten

Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine

CRISPR-Cas技术及其应用的进展：革新精准医学

Genome editing and plant transformation are crucial techniques in plant biotechnology, allowing for the precise modification of plant genomes to enhance agronomically essential traits. The advancement of CRISPR-based genome editing tools in plants is limited, among others, by developing novel in vitro tissue culture methodologies for efficient plant genetic transformation. In-planta methodologies offer a promising alternative to overcome tissue culture limitations and facilitate crops’ genetic improvement. The in-planta transformation methods can be categorized under the definition of means of plant genetic transformation with no or minimal tissue culture steps meeting the conditions for minimal steps: short duration with a limited number of transfers, high technical simplicity, limited list of hormones, and that the regeneration does not undergo callus development. In this review, we analyzed over 250 articles. We identified studies that follow an in-planta transformation methodology for delivering CRISPR/Cas9 components focusing on crop plants, as model species have been previously reviewed in detail. This approach has been successfully applied for genome editing in crop plants: camelina, cotton, lemon, melon, orange, peanut, rice, soybean, and wheat. Overall, this study underscores the importance of in-planta methodologies in overcoming the limitations of tissue culture and advancing the field of plant genome editing.

Alejandro Sebiani-Calvo

Alejandro Hernández-Soto

Götz Hensel

Andrés Gatica-Arias

Crop genome editing through tissue-culture-independent transformation methods

通过不依赖组织培养的转化方法进行作物基因组编辑

Huanglongbing (HLB) disease, caused by Candidatus Liberibacte r asiaticus ( Ca Las), severely impacts citrus production, and currently, there is no cure. Developing HLB-resistant or tolerant cultivars is crucial, with modifying defense-related genes being a promising approach to managing HLB. NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a positive regulator of systemic acquired resistance (SAR), which enhances resistance to pathogens, whereas NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 3 (NPR3) is a negative regulator of SAR. To unambiguously address the role of CsNPR3 in HLB, we introduced mutations into the CsNPR3 gene in sweet orange ( Citrus sinensis L. Osbeck) through genome editing and assessed their effects on morphology, physiology, and resistance/tolerance to HLB. Several genome-edited ‘Hamlin’ sweet orange trees harboring frameshift-inducing insertions or deletions were identified. After confirming the genome editing using Sanger sequencing, selected lines were grafted onto C-146 trifoliate hybrid rootstocks for clonal propagation. The progenies were then infected with Ca Las using a no-choice Asian Citrus Psyllid (ACP) feeding assay. Evaluation of the genetic and physiological characteristics of CsNPR3 -edited citrus trees under greenhouse conditions revealed that the edited trees exhibited greater vigor than the wild-type trees, despite the lack of significant differences in Ca Las titers. Although further field evaluation is needed, our findings indicate that CsNPR3 contributes to HLB-caused tree deterioration and demonstrate that editing CsNPR3 can enhance tolerance to HLB.

Trishna Tiwari

Cecile Robertson

Choaa El-Mohtar

Jude Grosser

Tripti Vashisth

Zhonglin Mou

Manjul Dutt

Genetic and physiological characteristics of CsNPR3 edited citrus and their impact on HLB tolerance

编辑柑橘CsNPR3的遗传和生理特性及其对黄龙病耐受性的影响

Large scale cancer genomic studies in patients have unveiled millions of non-coding variants. While a handful have been shown to drive cancer development, the vast majority have unknown function. This review describes the challenges of functionally annotating non-coding cancer variants and understanding how they contribute to cancer. We summarize recently developed high-throughput technologies to address these challenges. Finally, we outline future prospects for non-coding cancer genetics to help catalyze personalized cancer therapy.

Yihan Wang

Gary C. Hon

Towards functional maps of non-coding variants in cancer

癌症中非编码变异的功能图谱研究

短名	Front. Genome Ed.
Journal Impact	5.00
国际分区	GENETICS & HEREDITY(Q1)

ISSN	2673-3439
h-index	19

涉及主题	生物化学生物化学遗传学基因清脆的基因组基因组编辑计算生物学医学Cas9内科学DNA细胞生物学
出版信息	出版商: Frontiers Media SA，出版周期: ，期刊类型: journal，开源期刊: 是
基本数据	创刊年份: 2019，原创研究文献占比: 0.00%，自引率:8.20%， Gold OA占比: 100.00%
期刊费用	$2125详情
平均审稿周期	网友分享经验：13 Weeks

Frontiers in Genome Editing

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