Frontiers in genome editing最新文献_第3页

Epigenetic editing for autosomal dominant neurological disorders 表观遗传编辑治疗常染色体显性神经系统疾病

Frontiers in genome editing

Pub Date : 2024-03-06 DOI: 10.3389/fgeed.2024.1304110

J. Waldo, J. Halmai, Kyle D. Fink

Epigenetics refers to the molecules and mechanisms that modify gene expression states without changing the nucleotide context. These modifications are what encode the cell state during differentiation or epigenetic memory in mitosis. Epigenetic modifications can alter gene expression by changing the chromatin architecture by altering the affinity for DNA to wrap around histone octamers, forming nucleosomes. The higher affinity the DNA has for the histones, the tighter it will wrap and therefore induce a heterochromatin state, silencing gene expression. Several groups have shown the ability to harness the cell’s natural epigenetic modification pathways to engineer proteins that can induce changes in epigenetics and consequently regulate gene expression. Therefore, epigenetic modification can be used to target and treat disorders through the modification of endogenous gene expression. The use of epigenetic modifications may prove an effective path towards regulating gene expression to potentially correct or cure genetic disorders.

表观遗传学指的是在不改变核苷酸上下文的情况下改变基因表达状态的分子和机制。这些修饰编码了细胞在分化过程中的状态或有丝分裂过程中的表观遗传记忆。表观遗传修饰可通过改变染色质结构来改变基因表达，改变 DNA 与组蛋白八聚体的亲和力，形成核小体。DNA 与组蛋白的亲和力越高，就会缠绕得越紧，从而诱发异染色质状态，使基因表达沉默。一些研究小组已经证明，利用细胞的天然表观遗传修饰途径，可以设计出能够诱导表观遗传学变化的蛋白质，从而调节基因表达。因此，表观遗传修饰可用于通过改变内源性基因表达来靶向治疗疾病。使用表观遗传修饰可能被证明是调节基因表达以纠正或治疗遗传疾病的有效途径。

引用次数: 0

Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome 无核酸精确基因组编辑可纠正与雷特综合征相关的 MECP2 基因突变

Frontiers in genome editing

Pub Date : 2024-03-01 DOI: 10.3389/fgeed.2024.1346781

Swati Bijlani, Ka Ming Pang, Lakshmi V. Bugga, Sampath Rangasamy, Vinodh Narayanan, Saswati Chatterjee

Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5′ and 3′ ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.

雷特综合征是一种获得性进行性神经发育障碍，由 X 连锁 MECP2 基因的新突变引起。雷特综合征主要影响杂合子雌性患者，而受影响的男性半杂合子患者很少存活。Rett 综合征的基因治疗具有挑战性，因为需要严格的表达调控，过度表达或表达不足都会产生毒性。结合调控 miRNA 目标序列的 MECP2 异位表达取得了一些成功，但这种方法的持久性仍然未知。在这里，我们评估了一种基于无核酸酶同源重组（HR）的基因组编辑策略，以纠正MECP2基因的突变。干细胞衍生的AAVHSCs先前已被证明能介导无缝、精确的基于HR的基因组编辑。我们测试了基于HR的基因组编辑纠正MECP2基因第3和第4外显子中致病突变的能力，并在保留与MECP2表达相关的所有原生基因组调控元件的同时恢复野生型序列，从而有可能解决Rett综合征基因治疗中的一个重要问题。此外，由于突变是直接在基因组水平上进行编辑的，因此预计这种校正将是持久的，后代细胞将继承编辑后的基因。AAVHSC MECP2 编辑载体的设计与目标 MECP2 区域完全同源，并在外显子 4 的末端插入了无启动子的 Venus 报告基因。在一组外显子 3 和 4 发生突变的 Rett 细胞系中对 AAVHSC 编辑进行的评估表明，编辑后的 MECP2 基因的表达得到了成功的纠正和挽救。对编辑过的 Rett 细胞进行序列分析后发现，第 3 和第 4 外显子中的突变都得到了成功而准确的校正，并能绘制出 HR 交叉事件的图谱。只有当突变的5′端和3′端两侧都有交叉事件时，才能观察到成功的校正，而当两个交叉事件都完全发生在突变的上游或下游时，则不能观察到成功的校正。重要的是，我们得出结论，在分析的每个 Rett 株系中，致病突变都被成功纠正，这证明了基于 HR 的基因组编辑具有治疗潜力。

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引用次数: 0

Revolutionizing in vivo therapy with CRISPR/Cas genome editing: breakthroughs, opportunities and challenges. 利用 CRISPR/Cas 基因组编辑技术革新体内治疗：突破、机遇与挑战。

Frontiers in genome editing

Pub Date : 2024-02-01 eCollection Date: 2024-01-01 DOI: 10.3389/fgeed.2024.1342193

Arturo Macarrón Palacios, Patrick Korus, Bodo G C Wilkens, Najmeh Heshmatpour, Sarita R Patnaik

Genome editing using the CRISPR/Cas system has revolutionized the field of genetic engineering, offering unprecedented opportunities for therapeutic applications in vivo. Despite the numerous ongoing clinical trials focusing on ex vivo genome editing, recent studies emphasize the therapeutic promise of in vivo gene editing using CRISPR/Cas technology. However, it is worth noting that the complete attainment of the inherent capabilities of in vivo therapy in humans is yet to be accomplished. Before the full realization of in vivo therapeutic potential, it is crucial to achieve enhanced specificity in selectively targeting defective cells while minimizing harm to healthy cells. This review examines emerging studies, focusing on CRISPR/Cas-based pre-clinical and clinical trials for innovative therapeutic approaches for a wide range of diseases. Furthermore, we emphasize targeting cancer-specific sequences target in genes associated with tumors, shedding light on the diverse strategies employed in cancer treatment. We highlight the various challenges associated with in vivo CRISPR/Cas-based cancer therapy and explore their prospective clinical translatability and the strategies employed to overcome these obstacles.

使用 CRISPR/Cas 系统进行基因组编辑给基因工程领域带来了革命性的变化，为体内治疗应用提供了前所未有的机会。尽管目前正在进行的大量临床试验都以体内外基因组编辑为重点，但最近的研究强调了使用 CRISPR/Cas 技术进行体内基因编辑的治疗前景。不过，值得注意的是，在人类身上完全实现体内治疗的固有功能还有待时日。在完全实现体内治疗潜力之前，关键是要提高选择性靶向缺陷细胞的特异性，同时尽量减少对健康细胞的伤害。本综述探讨了新出现的研究，重点关注基于 CRISPR/Cas 的临床前和临床试验，以创新的治疗方法治疗各种疾病。此外，我们还强调了针对与肿瘤相关基因中癌症特异性序列的靶标，揭示了癌症治疗中采用的各种策略。我们强调了与基于 CRISPR/Cas 的体内癌症疗法相关的各种挑战，并探讨了这些疗法的临床转化前景以及克服这些障碍的策略。

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引用次数: 0

CRISPR-mediated promoter editing of a cis-regulatory element of OsNAS2 increases Zn uptake/translocation and plant yield in rice CRISPR 介导的 OsNAS2 顺式调控元件启动子编辑可提高水稻的锌吸收/转运能力和植物产量

Frontiers in genome editing

Pub Date : 2024-01-23 DOI: 10.3389/fgeed.2023.1308228

Yvonne Ludwig, C. Dueñas, E. Arcillas, Reena Jesusa Macalalad-Cabral, Ajay Kohli, Russell Reinke, I. Slamet-Loedin

Developing nutritious rice with a higher yield is one approach to alleviating the problem of micronutrient deficiency in developing countries, especially human malnutrition involving zinc and iron (Fe) deficiency, and achieving better adoption. The transport of micronutrients such as Fe and Zn is mainly regulated via the nicotianamine synthase (OsNAS) gene family, whereas yield is a complex trait that involves multiple loci. Genome editing via CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, focusing on the OsNAS2 promoter, particularly the deletion of the cis-regulatory element ARR1AT at position −933, was conducted for an enhanced accumulation of Zn in the grain and per plant. The results showed that our promoter editing increased Zn concentration per plant. Evidence also showed that an improved spikelet number per main panicle led to increased grain per plant. The traits were inherited in “transgene-free” and homozygous plant progenies. Further investigation needs to be conducted to validate trait performance under field conditions and elucidate the cause of the spikelet increase.

开发产量更高的营养水稻是缓解发展中国家微量营养素缺乏问题（尤其是涉及锌和铁（Fe）缺乏的人类营养不良问题）和实现更好采用的方法之一。铁和锌等微量营养元素的转运主要通过烟酰胺合成酶（OsNAS）基因家族调控，而产量则是一个涉及多个基因位点的复杂性状。为了提高谷粒和单株的锌积累，我们通过CRISPR（簇状规则间隔短回文重复）-Cas9对OsNAS2启动子进行了基因组编辑，特别是删除了位于-933位的顺式调控元件ARR1AT。结果表明，我们的启动子编辑增加了每株植物的锌浓度。证据还显示，每株主圆锥花序的小穗数增加也会导致单株籽粒增加。这些性状在 "无转基因 "和同源植物后代中得到了遗传。要验证田间条件下的性状表现并阐明小穗增加的原因，还需要进行进一步的调查。

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引用次数: 0

CRISPR/Cas9: an advanced platform for root and tuber crops improvement CRISPR/Cas9：改良块根和块茎作物的先进平台

Frontiers in genome editing

Pub Date : 2024-01-19 DOI: 10.3389/fgeed.2023.1242510

K. Divya, Makeshkumar Thangaraj, N. Krishna Radhika

Root and tuber crops (RTCs), which include cassava, potato, sweet potato, and yams, principally function as staple crops for a considerable fraction of the world population, in addition to their diverse applications in nutrition, industry, and bioenergy sectors. Even then, RTCs are an underutilized group considering their potential as industrial raw material. Complexities in conventional RTC improvement programs curb the extensive exploitation of the potentials of this group of crop species for food, energy production, value addition, and sustainable development. Now, with the advent of whole-genome sequencing, sufficient sequence data are available for cassava, sweet potato, and potato. These genomic resources provide enormous scope for the improvement of tuber crops, to make them better suited for agronomic and industrial applications. There has been remarkable progress in RTC improvement through the deployment of new strategies like gene editing over the last decade. This review brings out the major areas where CRISPR/Cas technology has improved tuber crops. Strategies for genetic transformation of RTCs with CRISPR/Cas9 constructs and regeneration of edited lines and the bottlenecks encountered in their establishment are also discussed. Certain attributes of tuber crops requiring focus in future research along with putative editing targets are also indicated. Altogether, this review provides a comprehensive account of developments achieved, future lines of research, bottlenecks, and major experimental concerns regarding the establishment of CRISPR/Cas9-based gene editing in RTCs.

块根和块茎作物（RTCs）包括木薯、马铃薯、甘薯和山药，除了在营养、工业和生物能源领域有多种用途外，主要还是世界上相当一部分人口的主食作物。即便如此，考虑到其作为工业原料的潜力，RTC 仍然是一类未得到充分利用的作物。传统 RTC 改良计划的复杂性阻碍了广泛开发这类作物在粮食、能源生产、增值和可持续发展方面的潜力。现在，随着全基因组测序技术的出现，木薯、甘薯和马铃薯已经有了足够的序列数据。这些基因组资源为改良块茎作物提供了巨大的空间，使其更适合农艺学和工业应用。过去十年中，通过采用基因编辑等新策略，在改良块茎作物方面取得了显著进展。本综述介绍了 CRISPR/Cas 技术改良块茎作物的主要领域。此外，还讨论了用 CRISPR/Cas9 构建物对 RTC 进行遗传转化和编辑品系再生的策略，以及在建立这些品系过程中遇到的瓶颈。此外，还指出了未来研究需要重点关注的块茎作物的某些特性以及可能的编辑目标。总之，本综述全面介绍了在 RTCs 中建立基于 CRISPR/Cas9 的基因编辑所取得的进展、未来的研究方向、瓶颈以及主要的实验问题。

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引用次数: 0

Editorial: Genome editing in stem cells 社论：干细胞中的基因组编辑

Frontiers in genome editing

Pub Date : 2024-01-17 DOI: 10.3389/fgeed.2024.1357369

Delger Bayarsaikhan, Edina Poletto

引用次数: 0

Computational analysis of cas proteins unlocks new potential in HIV-1 targeted gene therapy 对 Cas 蛋白的计算分析为 HIV-1 靶向基因疗法释放出新潜力

Frontiers in genome editing

Pub Date : 2024-01-04 DOI: 10.3389/fgeed.2023.1248982

W. Dampier, Rachel Berman, M. Nonnemacher, B. Wigdahl

Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro, ex vivo, and in vivo. These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability.Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential.Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV.Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors.

导言：随着抗逆转录病毒疗法（ART）的出现，人类免疫缺陷病毒 1 型（HIV-1）的流行速度有所减缓。然而，抗逆转录病毒疗法并不能治愈疾病，反而会使疾病发展成慢性感染。一种有希望治愈艾滋病的潜在策略是CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）/Cas基因编辑系统。最近的研究表明，该系统可以成功地编辑和/或切除感染细胞中的整合前病毒，并在体外、体内和体外抑制 HIV-1。这些研究主要是利用 SpCas9 或 SaCas9 进行的。然而，人们也经常发现其他的 Cas 蛋白，并对这些已知蛋白进行改造。替代的 Cas 分子对临近的原间隔基序（PAM）有不同的要求，这影响了 HIV-1 的可能靶区。对 Cas 蛋白或 gRNA 处理的其他修改会影响对 gRNA 与靶之间错配的容忍度。在降低脱靶风险的同时，这也影响了全面考虑 HIV-1 基因变异性的能力：本稿件试图利用一种计算方法来研究这些参数选择，以调查 Cas 编辑器是否适合用于 HIV-1 基因编辑。提名、多样化、缩小、筛选（NDNF）管道可衡量任何 PAM 潜在 gRNA 库的安全性、广泛性和有效性。这项技术用于评估 46 种不同的潜在 Cas 编辑器的艾滋病治疗潜力：我们的研究发现，更宽泛的 PAM 能够提高 SaCas9 和 LbCas12a 等编辑器的靶向潜力，具有更大的有用 gRNAs 库，而更宽泛的 PAM 则减少了 SpCas9 有用 gRNAs 库，但增加了可靶向位置的广度。对 Cas 编辑器错配耐受性的研究表明，2 个错配耐受性是靶上灵敏度和脱靶特异性之间的理想平衡。在所有被研究的 Cas 编辑器中，SpCas-NG 和 SPRY-Cas9 对 HIV 的总体安全、广泛和有效的 gRNA 数量最多：讨论：目前，较大的蛋白质和较宽的 PAM 可提高靶向能力。讨论：目前，更大的蛋白质和更宽的 PAMs 能带来更好的靶向能力，这意味着研究应着眼于提供更长的有效载荷，或增加目前可用的小型 Cas 编辑器的广度。随着更多 Cas 编辑器的发现和采用，HIV-1 基因编辑领域的研究人员必须探索更广阔的 Cas 编辑器世界。

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引用次数: 0

What do people think about genetic engineering? A systematic review of questionnaire surveys before and after the introduction of CRISPR 人们如何看待基因工程？对CRISPR问世前后问卷调查的系统回顾

Frontiers in genome editing

Pub Date : 2023-12-19 DOI: 10.3389/fgeed.2023.1284547

Pedro Dias Ramos, Maria Strecht Almeida, Ingrid Anna Sofia Olsson

The advent of CRISPR-Cas9 in 2012 started revolutionizing the field of genetics by broadening the access to a method for precise modification of the human genome. It also brought renewed attention to the ethical issues of genetic modification and the societal acceptance of technology for this purpose. So far, many surveys assessing public attitudes toward genetic modification have been conducted worldwide. Here, we present the results of a systematic review of primary publications of surveys addressing public attitudes toward genetic modification as well as the awareness and knowledge about the technology required for genetic modification. A total of 53 primary publications (1987–2020) focusing on applications in humans and non-human animals were identified, covering countries in four continents. Of the 53 studies, 30 studies from until and including 2012 (pre-CRISPR) address gene therapy in humans and genetic modification of animals for food production and biomedical research. The remaining 23 studies from after 2013 (CRISPR) address gene editing in humans and animals. Across countries, respondents see gene therapy for disease treatment or prevention in humans as desirable and highly acceptable, whereas enhancement is generally met with opposition. When the study distinguishes between somatic and germline applications, somatic gene editing is generally accepted, whereas germline applications are met with ambivalence. The purpose of the application is also important for assessing attitudes toward genetically modified animals: modification in food production is much less accepted than for biomedical application in pre-CRISPR studies. A relationship between knowledge/awareness and attitude toward genetic modification is often present. A critical appraisal of methodology quality in the primary publications with regards to sampling and questionnaire design, development, and administration shows that there is considerable scope for improvement in the reporting of methodological detail. Lack of information is more common in earlier studies, which probably reflects the changing practice in the field.

2012 年，CRISPR-Cas9 的问世拓宽了人类基因组精确改造方法的途径，开始了遗传学领域的一场革命。同时，它也使人们重新关注基因改造的伦理问题以及社会对基因改造技术的接受程度。迄今为止，世界范围内已经开展了许多调查，评估公众对基因改造的态度。在此，我们对公众对转基因的态度以及对转基因所需技术的认识和了解的调查的主要出版物进行了系统回顾。我们共鉴定了 53 篇主要出版物（1987-2020 年），重点关注人类和非人类动物的应用，涵盖四大洲的国家。在这 53 项研究中，有 30 项研究是在 2012 年（CRISPR 之前）之前（包括 2012 年）进行的，涉及人类基因疗法以及用于食品生产和生物医学研究的动物基因修饰。其余 23 项研究是在 2013 年（CRISPR）之后进行的，涉及人类和动物的基因编辑。在所有国家中，受访者都认为用于人类疾病治疗或预防的基因疗法是可取的，可接受度很高，而基因增强则普遍遭到反对。当研究对体细胞和种系应用进行区分时，体细胞基因编辑被普遍接受，而种系应用则受到矛盾的对待。应用的目的对于评估人们对转基因动物的态度也很重要：在CRISPR研究之前的研究中，食品生产中的转基因技术比生物医学应用中的转基因技术更难被接受。知识/意识与对转基因的态度之间往往存在关系。对主要出版物中有关抽样和问卷设计、开发和管理的方法质量进行的严格评估表明，在报告方法细节方面还有很大的改进余地。缺乏信息的情况在早期研究中更为常见，这可能反映了该领域实践的变化。

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引用次数: 0

Diversity of transgene integration and gene-editing events in wheat (Triticum aestivum L.) transgenic plants generated using Agrobacterium-mediated transformation. 利用农杆菌介导转化产生的小麦（Triticum aestivum L.）转基因植株中转基因整合和基因编辑事件的多样性。

Frontiers in genome editing

Pub Date : 2023-12-19 eCollection Date: 2023-01-01 DOI: 10.3389/fgeed.2023.1265103

Louie Cris Lopos, Natalia V Bykova, Janeen Robinson, Susan Brown, Kerry Ward, Andriy Bilichak

Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype-dependency. Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) chimeric gene. Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low- and high-copy-number integration events were recovered in the T₀ population with various levels of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome. A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary screening of transgene quality and GE events in the T₀ population of plants regenerated through the co-delivery of GRF-GIF can allow for the propagation of the best candidates for further phenotypic analysis.

通过基因编辑（GE）改善农作物的农艺性状有赖于高效的转化方案来传递 CRISPR/Cas9 编码的转基因。最近，一些与胚胎发生相关的基因被描述出来，这些基因的联合传递大大提高了转化效率，降低了基因型依赖性。在这里，我们描述了小麦（cv. Fielder）在转入生长调节因子 4（GRF4）及其辅助因子 GRF-INTERACTING FACTOR 1（GIF1）嵌合基因后的转基因和 GE 事件。在我们的实验中，转化效率从 22% 到 68%不等，而且编辑事件能忠实地传播到下一代。在 T0 群体中，低拷贝数和高拷贝数整合事件都得到了恢复，T-DNA 左右边界的完整性程度各不相同。我们还利用基因组中针对 30 个基因位点的 10 种不同 gRNA 生成了小麦植株群体。通过比较目标位点的表观遗传学特征和编辑效率，发现染色质可及性和诱变率之间存在显著的正相关。总之，在通过联合递送 GRF-GIF 再生的 T0 植株群体中对转基因质量和 GE 事件进行初步筛选，可以繁殖出最佳候选者，以便进一步进行表型分析。

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引用次数: 0

Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol 通过改进的形态发生调节剂辅助转化协议实现热带玉米品系的基因组编辑

Frontiers in genome editing

Pub Date : 2023-12-07 DOI: 10.3389/fgeed.2023.1241035

José Hernandes-Lopes, Maísa Siqueira Pinto, Letícia Rios Vieira, Patrícia Brant Monteiro, Sophia V. Gerasimova, Juliana Vieira Almeida Nonato, Maria Helena Faustinoni Bruno, Alexander Vikhorev, Fernanda Rausch-Fernandes, I. Gerhardt, L. Pauwels, Paulo Arruda, R. A. Dante, J. Yassitepe

The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve ’universal’ transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.

许多玉米(Zea mays)基因型表现出对传统遗传转化方案的抗拒，这对基因组编辑(GE)在这一主要作物物种中的大规模应用构成了重大挑战。虽然一些玉米基因型被广泛用于遗传转化，但它们被证明不适合在田间试验或商业应用中进行农艺试验。尽管热带地区的玉米产量占相当大的比例，但适应温带地区的可转化玉米品系的优势加剧了这一挑战。形态发生调控因子(MRs)的异位表达是克服低效率和基因型依赖的一种有前途的方法，旨在实现玉米的“通用”转化和转基因能力。在这里，我们报告了利用先前优化的B104温带自交系的基于mr的农杆菌介导转化方案，成功地对农艺相关的热带玉米品系进行了转基因。为此，我们使用了基于CRISPR/ cas9的构建，旨在敲除VIRESCENT YELLOW-LIKE (VYL)基因，从而产生易于识别的表型。在B104和3个热带品系制备的原生质体中证实了VYL突变，无论其中两个热带品系的VYL靶位点的种子区是否存在单核苷酸多态性(SNP)。五分之三的热带线路可以改造，效率高达6.63%。值得注意的是，97%的恢复事件在目标位点呈现索引，这些索引由下一代继承。我们观察到基于CRISPR/ cas9的构建体对VYL相似的VYL- modifier具有脱靶活性，这可能部分归因于WUSCHEL (WUS) MR.的表达。我们的研究结果证明了相关热带玉米品系的有效基因改造，扩大了这种主要作物基因型的可用性。

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引用次数: 0