CRISPR Journal最新文献_第6页

Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins. 通过DNA修复蛋白的组合融合提高Cas9介导的Knockin的准确性和效率。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 Epub Date: 2023-09-15 DOI: 10.1089/crispr.2023.0036

Ryan R Richardson, Marilyn Steyert, Saovleak N Khim, Garrett W Crutcher, Cheryl Brandenburg, Colin D Robertson, Andrea J Romanowski, Jeffrey Inen, Bekir Altas, Alexandros Poulopoulos

{"title":"Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins.","authors":"Ryan R Richardson, Marilyn Steyert, Saovleak N Khim, Garrett W Crutcher, Cheryl Brandenburg, Colin D Robertson, Andrea J Romanowski, Jeffrey Inen, Bekir Altas, Alexandros Poulopoulos","doi":"10.1089/crispr.2023.0036","DOIUrl":"10.1089/crispr.2023.0036","url":null,"abstract":"Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP[HE], increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10611978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10609434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels. 评估双核核苷酸编辑水平的APOBEC报告系统。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 Epub Date: 2023-09-06 DOI: 10.1089/crispr.2023.0027

Amanda E Rieffer, Yanjun Chen, Daniel J Salamango, Sofia N Moraes, Reuben S Harris

{"title":"APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels.","authors":"Amanda E Rieffer, Yanjun Chen, Daniel J Salamango, Sofia N Moraes, Reuben S Harris","doi":"10.1089/crispr.2023.0027","DOIUrl":"10.1089/crispr.2023.0027","url":null,"abstract":"Precision genome editing has become a reality with the discovery of base editors. Cytosine base editor (CBE) technologies are improving rapidly but are mostly optimized for TC dinucleotide targets. Here, we report the development and implementation of APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels (ARSENEL) in living cells. The ARSENEL panel is comprised of four constructs that quantitatively report editing of each of the four dinucleotide motifs (AC/CC/GC/TC) through real-time accumulation of eGFP fluorescence. Editing rates of APOBEC3Bctd and AIDΔC CBEs reflect established mechanistic preferences with intrinsic biases to TC and GC, respectively. Twelve different (new and established) base editors are tested here using this system with a full-length APOBEC3B CBE showing the greatest on-target TC specificity and an APOBEC3A construct showing the highest editing efficiency. In addition, ARSENEL enables real-time assessment of natural and synthetic APOBEC inhibitors with the most potent to-date being the large subunit of the Epstein-Barr virus ribonucleotide reductase. These reporters have the potential to play important roles in research and development as precision genome engineering technologies progress toward achieving maximal specificity and efficiency.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10611974/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10168892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Special Issue: CRISPR Trials. 特刊：CRISPR试验。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 DOI: 10.1089/crispr.2023.29166.cfp

Fyodor Urnov

引用次数: 0

Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection. 基于Cas12a的扩增游离核酸检测研究进展。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 Epub Date: 2023-09-25 DOI: 10.1089/crispr.2023.0023

Shixin Ji, Xueli Wang, Yangkun Wang, Yingqi Sun, Yingying Su, Xiaosong Lv, Xiangwei Song

{"title":"Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection.","authors":"Shixin Ji, Xueli Wang, Yangkun Wang, Yingqi Sun, Yingying Su, Xiaosong Lv, Xiangwei Song","doi":"10.1089/crispr.2023.0023","DOIUrl":"10.1089/crispr.2023.0023","url":null,"abstract":"In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have been widely used in nucleic acid detection, especially the CRISPR-Cas12a system, which can trans cleave single-stranded DNA and can realize the detection of DNA targets. But, amplification of nucleic acids is still required to further improve detection sensitivity, which makes Cas12a-based amplification-free nucleic acid detection methods a great challenge. This article reviews the recent progress of Cas12a-based amplification-free detection methods for nucleic acids. These detection methods apply electrochemical detection methods, fluorescence detection methods, noble metal nanomaterial detection methods, and lateral flow assay. Under various optimization strategies, unamplified nucleic acids have the same sensitivity as amplified nucleic acids. At the same time, the article discusses the advantages and disadvantages of each method and further discusses the current challenges such as off-target effects and the ability to achieve high-throughput detection. Amplification-free nucleic acid detection technology based on CRISPR-Cas12a has great potential in the biomedical field.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41140397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae. 测量遗传异质性和染色体反转对CRISPR-Cas9基因驱动在不同冈比亚按蚊菌株中的效力的影响。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 Epub Date: 2023-09-13 DOI: 10.1089/crispr.2023.0029

Poppy Pescod, Giulia Bevivino, Amalia Anthousi, Ruth Shelton, Josephine Shepherd, Fabrizio Lombardo, Tony Nolan

{"title":"Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae.","authors":"Poppy Pescod, Giulia Bevivino, Amalia Anthousi, Ruth Shelton, Josephine Shepherd, Fabrizio Lombardo, Tony Nolan","doi":"10.1089/crispr.2023.0029","DOIUrl":"10.1089/crispr.2023.0029","url":null,"abstract":"The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site (\"homing\"), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of laboratory-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10215382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE): An Experimental and Computational Approach for Analyzing CRISPR-Edited Cells. CRISPR定位编辑的基因分型多重测序（GMUSCLE）：一种分析CRISPR编辑细胞的实验和计算方法。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 DOI: 10.1089/crispr.2023.0021

Peng Zhang, Laurent Abel, Jean-Laurent Casanova, Rui Yang

{"title":"Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE): An Experimental and Computational Approach for Analyzing CRISPR-Edited Cells.","authors":"Peng Zhang, Laurent Abel, Jean-Laurent Casanova, Rui Yang","doi":"10.1089/crispr.2023.0021","DOIUrl":"10.1089/crispr.2023.0021","url":null,"abstract":"Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) creates double-stranded breaks, the repair of which generates indels around the target sites. These repairs can be mono-/multi-allelic, and the editing is often random and sometimes prolonged, resulting in considerable intercellular heterogeneity. The genotyping of CRISPR-Cas9-edited cells is challenging and the traditional genotyping methods are laborious. We introduce here a streamlined experimental and computational protocol for genotyping CRISPR-Cas9 genome-edited cells including cost-effective multiplexed sequencing and the software Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE). In this approach, CRISPR-Cas9-edited products are sequenced in great depth, then GMUSCLE quantitatively and qualitatively identifies the genotypes, which enable the selection and investigation of cell clones with genotypes of interest. We validate the protocol and software by performing CRISPR-Cas9-mediated disruption on interferon-α/β receptor alpha, multiplexed sequencing, and identifying the genotypes simultaneously for 20 cell clones. Besides the multiplexed sequencing ability of this protocol, GMUSCLE is also applicable for the sequencing data from bulk cell populations. GMUSCLE is publicly available at our HGIDSOFT server and GitHub.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10611965/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41220060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The CRISPR Toolbox: The End of the Beginning. CRISPR工具箱：开始的结束。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 DOI: 10.1089/crispr.2023.29167.editorial

Rodolphe Barrangou

引用次数: 0

Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells. 曲霉菌素A用于人多能干细胞的有效CRISPR-Cas9基因编辑。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-10-01 Epub Date: 2023-09-07 DOI: 10.1089/crispr.2023.0033

Kaivalya Molugu, Namita Khajanchi, Cicera R Lazzarotto, Shengdar Q Tsai, Krishanu Saha

引用次数: 0

Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2. 泛冠状病毒CRISPR-CasRx效应系统显著降低HCoV-OC43、HCoV-229E和SARS-CoV-2的活价

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-08-01 DOI: 10.1089/crispr.2022.0095

Cathryn M Mayes, Joshua L Santarpia

{"title":"Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.","authors":"Cathryn M Mayes, Joshua L Santarpia","doi":"10.1089/crispr.2022.0095","DOIUrl":"https://doi.org/10.1089/crispr.2022.0095","url":null,"abstract":"CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10457650/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10152767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

My CRISPR Story: Back to Brazil. 我的CRISPR故事:回到巴西。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2023-08-01 DOI: 10.1089/crispr.2023.0032

Daniel F M Monte

引用次数: 0