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Special Issue: CRISPR Trials. 特刊:CRISPR 试验。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 DOI: 10.1089/crispr.2023.29166.cfp2
Fyodor Urnov
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引用次数: 0
The Expanding Dissemination and Distribution Patterns of Diverse CRISPR Plasmids by Addgene. 利用Addgene扩大CRISPR质粒的传播和分布模式。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 Epub Date: 2023-11-22 DOI: 10.1089/crispr.2023.0059
Brook Pyhtila, Seth Kasowitz, Rachel Leeson, Rodolphe Barrangou

CRISPR-based technologies have rapidly enabled the democratization of genome editing in academic institutions through distribution by Addgene over the past decade. Recently, several distribution milestones have been reached, with a collection of >15,000 plasmids deposited by >1,000 laboratories spanning ∼40 countries now shipped 300,000 times to ∼5,000 organizations traversing ∼100 countries. Yet, both deposits of and requests for CRISPR plasmids continue to rise for this disruptive technology. Distribution patterns revealed robust demand for three distinct classes of CRISPR effectors, namely nucleases (e.g., Cas9 and Cas12), modulators (deactivated CRISPR nucleases fused to transcriptional regulators and epigenome modifiers), and chimeric effectors (Cas proteins fused to enzymes carrying out other activities such as deamination, reverse transcription, transposition, and integration). Yearly deposits over the past decade are requested in near-even proportions, reflecting continuous technological development and requests for novel constructs. Though it is unclear whether the slowing rate of requests is inherent to a pandemic operational lag or a transition from emerging to mature technology, it is noteworthy that the relative proportion of requests from plasmids deposited in the previous year remains stable, suggesting robust development of novel tools concurrent with continued adoption of editing, base editing, prime editing, and more. Predictably, most requested plasmids are designed for mammalian genome manipulation, presumably for medical research and human health pursuits, reflecting investments in therapeutic applications. Concurrently, requests for plant and microbial constructs are on the rise, especially in regions of the world more reliant on local agricultural inputs and focused on food and feed applications, illustrating continued diversification of genome editing applications.

在过去的十年里,通过Addgene的分销,基于crispr的技术迅速实现了学术机构基因组编辑的民主化。最近,已经达到了几个分销里程碑,跨越~ 40个国家的>1,000个实验室存放的>15,000个质粒现已运送30万次到跨越~ 100个国家的~ 5,000个组织。然而,对CRISPR质粒的储备和需求都在继续增加,因为这项颠覆性的技术。分布模式显示了对三种不同类型的CRISPR效应物的强劲需求,即核酸酶(例如Cas9和Cas12),调节剂(失活的CRISPR核酸酶融合到转录调节剂和表观基因组修饰剂中)和嵌合效应物(Cas蛋白融合到执行其他活动的酶中,如脱氨、逆转录、转位和整合)。在过去的十年中,每年的存款要求几乎均匀,反映了持续的技术发展和对新结构的要求。虽然尚不清楚请求速度放缓是大流行操作滞后还是新兴技术向成熟技术的过渡所固有的,但值得注意的是,上一年沉积的质粒请求的相对比例保持稳定,这表明在继续采用编辑、碱基编辑、主要编辑等方法的同时,新工具的强劲发展。可以预见的是,大多数要求的质粒是为哺乳动物基因组操作而设计的,可能是为了医学研究和人类健康追求,反映了对治疗应用的投资。与此同时,对植物和微生物构建物的需求也在增加,特别是在世界上更依赖当地农业投入并侧重于食品和饲料应用的地区,这表明基因组编辑应用的持续多样化。
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引用次数: 0
Engineered Antiviral Sensor Targets Infected Mosquitoes. 工程抗病毒传感器锁定受感染的蚊子
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 DOI: 10.1089/crispr.2023.0056
Elena Dalla Benetta, Adam J López-Denman, Hsing-Han Li, Reem A Masri, Daniel J Brogan, Michelle Bui, Ting Yang, Ming Li, Michael Dunn, Melissa J Klein, Sarah Jackson, Kyle Catalan, Kim R Blasdell, Priscilla Tng, Igor Antoshechkin, Luke S Alphey, Prasad N Paradkar, Omar S Akbari

Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

病媒疾病负担的不断增加对全球健康构成了重大风险,因此针对蚊子的创新工具至关重要。CRISPR-Cas 技术在开发用于操纵各种真核生物基因组的强大工具方面发挥了至关重要的作用。虽然大量工作集中在利用第二类第二型 CRISPR-Cas9 系统进行 DNA 靶向,但这些模式无法靶向 RNA 分子,限制了它们在对付 RNA 病毒方面的作用。最近,Cas13 家族作为一种有效的 RNA 靶向工具出现了;然而,这种技术在蚊子(尤其是埃及伊蚊)中的应用尚未完全实现。在这项研究中,我们设计了一种名为 REAPER(vRNA 表达激活有毒效应核糖核酸酶)的抗病毒策略,它利用了 CRISPR-Cas13 的可编程 RNA 靶向能力及其强大的附带活性。REAPER 隐藏在蚊子体内,直到吸食了有传染性的血食。一旦感染目标病毒 RNA,REAPER 就会激活,触发对目标虫媒病毒(如基孔肯雅病毒)的程序性破坏。因此,Cas13 介导的 RNA 靶向能显著减少病毒复制和病毒感染流行,其杂乱的附带活性甚至能在几天内杀死受感染的蚊子。这项创新的 REAPER 技术为抗击蚊虫病毒传播的有效分子遗传工具库增添了新的成员。
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引用次数: 0
CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa. CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2023-12-01 DOI: 10.1089/crispr.2023.0039
Erin R Burnight, Luke A Wiley, Nathaniel K Mullin, Malavika K Adur, Mallory J Lang, Cathryn M Cranston, Chunhua Jiao, Stephen R Russell, Elliot H Sohn, Ian C Han, Jason W Ross, Edwin M Stone, Robert F Mullins, Budd A Tucker

Rhodopsin (RHO) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOpCRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

Pro23His 等视黄素(RHO)突变是北美显性遗传性视网膜色素变性症的主要病因。与其他显性视网膜营养不良症一样,这些突变导致产生有毒的蛋白质产物,治疗需要敲除突变等位基因。本研究的目的是开发一种 CRISPR-Cas9 介导的转录抑制策略,使用的是与 Krüppel-associated box(KRAB)转录抑制结构域融合的催化活性金黄色葡萄球菌 Cas9(dCas9)。利用克隆在 RHO 启动子片段(核苷酸 -1403 至 +73)下游的携带绿色荧光蛋白(GFP)的报告基因构建物,我们证明 RHOpCRISPRi 处理的 RHO 启动子活性比仅用质粒的对照组降低了 74-84%。在对人类视网膜外植体和转基因 Pro23His 突变体猪进行视网膜下转导后,视网膜上的视紫红质蛋白被显著敲除。体内突变转基因的抑制与内质网(ER)应激和细胞凋亡标记物的减少以及感光细胞层厚度的保持有关。
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引用次数: 0
A Fluorescent Reporter Mouse for In Vivo Assessment of Genome Editing with Diverse Cas Nucleases and Prime Editors. 一种荧光报告小鼠,用于在体内评估使用多种 Cas 核酸酶和主编辑器进行基因组编辑的效果。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2023-12-01 DOI: 10.1089/crispr.2023.0048
Zexiang Chen, Suet-Yan Kwan, Aamir Mir, Max Hazeltine, Minwook Shin, Shun-Qing Liang, Io Long Chan, Karen Kelly, Krishna S Ghanta, Nicholas Gaston, Yueying Cao, Jun Xie, Guangping Gao, Wen Xue, Erik J Sontheimer, Jonathan K Watts

CRISPR-based genome-editing technologies, including nuclease editing, base editing, and prime editing, have recently revolutionized the development of therapeutics targeting disease-causing mutations. To advance the assessment and development of genome editing tools, a robust mouse model is valuable, particularly for evaluating in vivo activity and delivery strategies. In this study, we successfully generated a knock-in mouse line carrying the Traffic Light Reporter design known as TLR-multi-Cas variant 1 (TLR-MCV1). We comprehensively validated the functionality of this mouse model for both in vitro and in vivo nuclease and prime editing. The TLR-MCV1 reporter mouse represents a versatile and powerful tool for expediting the development of editing technologies and their therapeutic applications.

基于 CRISPR 的基因组编辑技术(包括核酸酶编辑、碱基编辑和质粒编辑)最近彻底改变了针对致病突变的疗法的开发。为了推动基因组编辑工具的评估和开发,一个强大的小鼠模型是非常有价值的,尤其是在评估体内活性和递送策略方面。在这项研究中,我们成功培育出了一种携带交通灯报告设计的基因敲入小鼠品系,即 TLR-multi-Cas变体 1(TLR-MCV1)。我们全面验证了这种小鼠模型在体外和体内核酸酶和质粒编辑方面的功能。TLR-MCV1 报告小鼠是一种多功能的强大工具,可加快编辑技术的开发及其治疗应用。
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引用次数: 0
First Drug Approval Rises the CRISPR Tide. 首次药物批准掀起 CRISPR 浪潮。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 Epub Date: 2023-12-05 DOI: 10.1089/crispr.2023.29168.editorial
Rodolphe Barrangou
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引用次数: 0
Correction to: APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels by Rieffer et al. The CRISPR Journal, 2023;6(5):430-446; DOI: 10.1089/crispr.2023.0027. Rieffer等人对《评估双核核苷酸编辑水平的APOBEC报告系统》的更正。CRISPR期刊,2023;6(5):430-446;DOI:10.1089/crispr.2023.0027。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 Epub Date: 2023-11-08 DOI: 10.1089/crispr.2023.0027.correx
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引用次数: 0
Modified Cas9-Guided Oxford Nanopore Technology Sequencing Uncovers Single and Multiple Transgene Insertion Sites in a Zebrafish Melanoma Model. 改良 Cas9 引导的牛津纳米孔技术测序发现斑马鱼黑色素瘤模型中的单个和多个转基因插入位点。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 DOI: 10.1089/crispr.2023.0062
Raffaella De Paolo, Uday Munagala, Francesco Cucco, Samanta Sarti, Letizia Pitto, Filippo Martignano, Silvestro G Conticello, Laura Poliseno
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引用次数: 0
A PCR-Induced Mutagenesis-Restriction Fragment Length Polymorphism Method for the Detection of CRISPR-Induced Indels. 用于检测 CRISPR 诱导的 Indels 的 PCR 诱导突变-限制性片段长度多态性方法。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 Epub Date: 2023-12-05 DOI: 10.1089/crispr.2023.0047
Lydia Angelopoulou, Electra Stylianopoulou, Konstantinos Tegopoulos, Ioanna Farmakioti, Maria Grigoriou, George Skavdis

As CRISPR-based technologies are widely used for knocking out genes in cell lines and organisms, there is a need for the development of reliable, cost-effective, and fast methods that identify fully mutated clones. In this context, we present a novel strategy named PCR-induced mutagenesis-restriction fragment length polymorphism (PIM-RFLP), which is based on the well-documented robustness and simplicity of the classical PCR-RFLP approach. PIM-RFLP allows the assessment of the editing efficiency in pools of edited cells and the effective identification of fully mutated single-cell clones. It is based on the creation by mutagenic PCR of a restriction enzyme degenerate cleavage site in the PCR product of the wild-type allele, which can then be distinguished from the indel-containing alleles following the standard RFLP procedure. PIM-RFLP is highly accessible, can be executed in a single day, and appears to outperform Sanger sequencing deconvolution algorithms in the detection of fully mutated clones.

由于基于 CRISPR 的技术被广泛用于敲除细胞系和生物体中的基因,因此需要开发可靠、经济、快速的方法来鉴定完全突变的克隆。在此背景下,我们提出了一种名为 "PCR诱导突变-限制性片段长度多态性(PIM-RFLP)"的新策略。PIM-RFLP 可以评估编辑细胞池的编辑效率,并有效鉴定完全突变的单细胞克隆。它的基础是通过诱变 PCR 在野生型等位基因的 PCR 产物中创建一个限制性酶变性裂解位点,然后按照标准 RFLP 程序将其与含 indel 的等位基因区分开来。PIM-RFLP 非常容易获得,可在一天内完成,而且在检测完全突变的克隆方面似乎优于 Sanger 测序解旋算法。
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引用次数: 0
Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage. SpyCas9的RuvC活性位点与二价金属的不同结合导致了非特异性DNA裂解。
IF 3.7 4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2023-12-01 DOI: 10.1089/crispr.2023.0022
Sydney N Newsom, Duen-Shian Wang, Saadi Rostami, Isabelle Schuster, Hari Priya Parameshwaran, Yadin G Joseph, Peter Z Qin, Jin Liu, Rakhi Rajan

To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9H982A) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn2+-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn2+, but not Mg2+, produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.

为了抵御移动遗传元件(MGEs),一些细菌和古细菌拥有聚类规则间隔短回文重复序列-CRISPR相关(CRISPR-Cas)适应性免疫系统。与 Cas 核酸酶结合的 CRISPR RNA(crRNA)根据序列互补性与 MGE 杂交,引导核酸酶裂解 MGE。这种可编程的 DNA 切割已被用于基因编辑。基因编辑常用的Cas核酸酶是化脓性链球菌Cas9(SpyCas9)。我们开发了一种不含 gRNA 的 SpyCas9 变体(SpyCas9H982A),它不含 gRNA,但对靶上裂解有更高的选择性。金属依赖性 RuvC 活性位点中的 H982A 取代可将 Mn2+ 依赖性无 gRNA DNA 切裂降低 167 倍。机理分子动力学分析表明,Mn2+(而非 Mg2+)产生的无 gRNA DNA 裂解能力状态会被 H982A 取代所破坏。我们的研究证明了通过调节阳离子与蛋白质的相互作用来设计更安全的基因编辑工具的可行性。
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引用次数: 0
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CRISPR Journal
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