CRISPR Journal最新文献

{"title":"The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity.","authors":"Suchita P Nety, Han Altae-Tran, Soumya Kannan, F Esra Demircioglu, Guilhem Faure, Seiichi Hirano, Kepler Mears, Yugang Zhang, Rhiannon K Macrae, Feng Zhang","doi":"10.1089/crispr.2023.0015","DOIUrl":"10.1089/crispr.2023.0015","url":null,"abstract":"<p><p>TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using <i>in vitro</i> purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA through 5' processing. We also uncover a potential <i>cis</i>-regulatory mechanism whereby a region of the TnpB mRNA inhibits DNA cleavage by the RNP complex. We further expand the characterization of TnpB by examining ωRNA processing and RNA-guided nuclease activity in 59 orthologs spanning the natural diversity of the TnpB family. This work reveals a new functionality, ωRNA biogenesis, of TnpB, and characterizes additional members of this biotechnologically useful family of programmable enzymes.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"232-242"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10278001/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9671763","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}

{"title":"Search for Origins of Anti-CRISPR Proteins by Structure Comparison.","authors":"Harutyun Sahakyan,&nbsp;Kira S Makarova,&nbsp;Eugene V Koonin","doi":"10.1089/crispr.2023.0011","DOIUrl":"https://doi.org/10.1089/crispr.2023.0011","url":null,"abstract":"<p><p>Many bacterial and archaeal viruses encode anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas systems via various mechanisms. The majority of the Acrs are small, non-enzymatic proteins that abrogate CRISPR activity by binding to Cas effector proteins. The Acrs evolve fast, due to the arms race with the respective CRISPR-Cas systems, which hampers the elucidation of their evolutionary origins by sequence comparison. We performed comprehensive structural modeling using AlphaFold2 for 3693 experimentally characterized and predicted Acrs, followed by a comparison to the protein structures in the Protein Data Bank database. After clustering the Acrs by sequence similarity, 363 high-quality structural models were obtained that accounted for 102 Acr families. Structure comparisons allowed the identification of homologs for 13 of these families that could be ancestors of the Acrs. Despite the limited extent of structural conservation, the inferred origins of Acrs show distinct trends, in particular, recruitment of toxins and antitoxins and SOS repair system components for the Acr function.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"222-231"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10285414/pdf/crispr.2023.0011.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9705259","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}

{"title":"Clarifying CRISPR: Why Repeats Identified in the Human Genome Should Not Be Considered CRISPRs.","authors":"Murat Buyukyoruk, William S Henriques, Blake Wiedenheft","doi":"10.1089/crispr.2022.0106","DOIUrl":"10.1089/crispr.2022.0106","url":null,"abstract":"<p><p>Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (<i>cas</i>) are essential components of adaptive immune systems that protect bacteria and archaea from viral infection. CRISPR-Cas systems are found in about 40% of bacterial and 85% of archaeal genomes, but not in eukaryotic genomes. Recently, an article published in <i>Communications Biology</i> reported the identification of 12,572 putative CRISPRs in the human genome, which they call \"hCRISPR.\" In this study, we attempt to reproduce this analysis and show that repetitive elements identified as putative CRISPR loci in the human genome contain neither the repeat-spacer-repeat architecture nor the <i>cas</i> genes characteristic of functional CRISPR systems.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"216-221"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10277986/pdf/crispr.2022.0106.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9679580","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}

{"title":"Correction to: <i>Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic</i> by Panda et al. <i>The CRISPR Journal</i>, 2023;6(2):176-182; DOI: 10.1089/crispr.2022.0050.","authors":"","doi":"10.1089/crispr.2022.0050.correx","DOIUrl":"https://doi.org/10.1089/crispr.2022.0050.correx","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"302"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10398720/pdf/crispr.2022.0050.correx.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9938137","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}

{"title":"<i>Special Issue</i>: Manipulating the Microbiome with CRISPR.","authors":"Brady Cress,&nbsp;Rodolphe Barrangou","doi":"10.1089/crispr.2023.29159.cfp2","DOIUrl":"https://doi.org/10.1089/crispr.2023.29159.cfp2","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"185"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9584363","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}

{"title":"Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.","authors":"Rebecca C Lamothe,&nbsp;Meghan D Storlie,&nbsp;Diego A Espinosa,&nbsp;Rachel Rudlaff,&nbsp;Patrick Browne,&nbsp;Jason Liu,&nbsp;Andres Rivas,&nbsp;Audra Devoto,&nbsp;Jennifer Oki,&nbsp;Ashcon Khoubyari,&nbsp;Daniela S Aliaga Goltsman,&nbsp;Jyun-Liang Lin,&nbsp;Cristina N Butterfield,&nbsp;Christopher T Brown,&nbsp;Brian C Thomas,&nbsp;Gregory J Cost","doi":"10.1089/crispr.2022.0089","DOIUrl":"https://doi.org/10.1089/crispr.2022.0089","url":null,"abstract":"<p><p>Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of β2-microglobulin, <i>TIGIT</i>, <i>FAS</i>, and <i>PDCD1</i>. Simultaneous double knockout of <i>TRAC</i> and <i>TRBC</i> was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into <i>TRAC</i> (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"243-260"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10277994/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9734046","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}

{"title":"Model System to Analyze RNA-Mediated DNA Repair in Mammalian Cells.","authors":"Lisa Tschage,&nbsp;Eric Kowarz,&nbsp;Rolf Marschalek","doi":"10.1089/crispr.2022.0105","DOIUrl":"https://doi.org/10.1089/crispr.2022.0105","url":null,"abstract":"<p><p>\"RNA-templated/directed DNA repair\" is a biological mechanism that has been experimentally demonstrated in bacteria, yeast, and mammalian cells. Recent study has shown that small noncoding RNAs (DDRNAs) and/or newly RNAPII transcribed RNAs (dilncRNAs) are orchestrating the initial steps of double-strand break (DSB) repair. In this study, we demonstrate that also pre-mRNA could be used as direct or indirect substrate for DSB repair. Our test system is based on (1) a stably integrated mutant reporter gene that produces constitutively a nonspliceable pre-mRNA, (2) a transiently expressed sgRNA-guided dCas13b::ADAR fusion protein to specifically RNA edit the nonspliceable pre-mRNA, and (3) transiently expressed <i>I-Sc</i>eI to create a DSB situation to study the effect of spliceable pre-mRNA on DNA repair. Based on our data, the RNA-edited pre-mRNA was used <i>in cis</i> for the DSB repair process, thereby converting the genomically encoded mutant reporter gene into an active reporter gene. Overexpression and knockdown of several cellular proteins were performed to delineate their role in this novel \"RNA-mediated end joining\" pathway.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"289-301"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9678200","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}

{"title":"Curing \"GFP-itis\" in Bacteria with Base Editors: Development of a Genome Editing Science Program Implemented with High School Biology Students.","authors":"Carlos A Vasquez, Mallory Evanoff, Brodie L Ranzau, Sifeng Gu, Emma Deters, Alexis C Komor","doi":"10.1089/crispr.2023.0002","DOIUrl":"10.1089/crispr.2023.0002","url":null,"abstract":"<p><p>The flexibility and precision of CRISPR-Cas9 and related technologies have made these genome editing tools increasingly popular in agriculture, medicine, and basic science research for the past decade. Genome editing will continue to be relevant and utilized across diverse scientific fields in the future. Given this, students should be introduced to genome editing technologies and encouraged to consider their ethical implications early on in precollege biology curricula. Furthermore, instruction on this topic presents an opportunity to create partnerships between researchers and educators at the K-12 levels that can strengthen student engagement in science, technology, engineering, and mathematics. To this end, we present a 3-day student-centered learning program to introduce high school students to genome editing technologies through a hands-on base editing experiment in <i>Escherichia coli</i>, accompanied by a relevant background lecture and facilitated ethics discussion. This unique partnership aims to educate students and provides a framework for research institutions to implement genome editing outreach programs at local high schools. We have included all requisite materials, including lecture slides, worksheets, experimental protocols, and suggestions on active learning strategies for others to reproduce our program with their local communities.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"186-195"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10277996/pdf/crispr.2023.0002.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9671496","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}

{"title":"CRISPR Conventions in a Polarized Era.","authors":"Rodolphe Barrangou","doi":"10.1089/crispr.2023.29160.editorial","DOIUrl":"https://doi.org/10.1089/crispr.2023.29160.editorial","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"183-184"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9672507","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}

{"title":"Efficient Genome and Base Editing in Human Cells Using ThermoCas9.","authors":"Despoina Trasanidou, Patrick Barendse, Evgenios Bouzetos, Laura de Haan, Hans Bouwmeester, Raymond H J Staals, Ioannis Mougiakos, John van der Oost","doi":"10.1089/crispr.2023.0005","DOIUrl":"10.1089/crispr.2023.0005","url":null,"abstract":"<p><p>Most genetic engineering applications reported thus far rely on the type II-A CRISPR-Cas9 nuclease from <i>Streptococcus pyogenes</i> (SpyCas9), limiting the genome-targeting scope. In this study, we demonstrate that a small, naturally accurate, and thermostable type II-C Cas9 ortholog from <i>Geobacillus thermodenitrificans</i> (ThermoCas9) with alternative target site preference is active in human cells, and it can be used as an efficient genome editing tool, especially for gene disruption. In addition, we develop a ThermoCas9-mediated base editor, called ThermoBE4, for programmable nicking and subsequent C-to-T conversions in human genomes. ThermoBE4 exhibits a three times larger window of activity compared with the corresponding SpyCas9 base editor (BE4), which may be an advantage for gene mutagenesis applications. Hence, ThermoCas9 provides an alternative platform that expands the targeting scope of both genome and base editing in human cells.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 3","pages":"278-288"},"PeriodicalIF":3.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9679606","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}