Genome editing technology and applications with the type I CRISPR system

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which are representative genome editing technologies, are classified into class 1 and class 2 in terms of evolutionary biology and are further classified into several subtypes. Class 2 CRISPR systems, including type II Cas9 and type V Cas12a, are the most commonly used for genome editing in eukaryotic cells, while type I CRISPR systems within Class 1 are also becoming available. Type I CRISPR recognizes longer target sequences than CRISPR-Cas9 and can induce large deletion mutations of several kilobases. These features demonstrate its potential as a novel and unique genome editing tool that can induce genetic disruption safely and reliably. Thus, it is expected to be utilized for gene therapy and industrial applications. Recently, the DNA cleavage mechanism of type I CRISPR has also revealed details from protein-complex analyses with X-ray crystallography, cryo-electron microscopy, and high-speed atomic force microscopy. The single-strand DNA trans-cleavage activity of type I CRISPR, called collateral activity, has broadened the potential application for CRISPR diagnostics, especially in the development of point-of-care testing methods for COVID-19. In this review, we present an overview of the type I CRISPR system, its application to genome editing, and genetic diagnosis using CRISPR-Cas3.Copyright © 2022

Stable production of recombinant SARS-CoV-2 receptor-binding domain in mammalian cells with co-expression of a fluorescent reporter and its validation as antigenic target for COVID-19 serology testing.

SARS-CoV-2 receptor binding domain (RBD) recognizes the angiotensin converting enzyme 2 (ACE2) receptor in host cells that enables infection. Due to its antigenic specificity, RBD production is important for development of serological assays. Here we have established a system for stable RBD production in HEK 293T mammalian cells that simultaneously express the recombinant fluorescent protein dTomato, which enables kinetic monitoring of RBD expression by fluorescence microscopy. In addition, we have validated the use of this recombinant RBD in an ELISA assay for the detection of anti-RBD antibodies in serum samples of COVID-19 convalescent patients. Recombinant RBD generated using this approach can be useful for generation of antibody-based therapeutics against SARS-CoV-2, as well serological assays aimed to test antibody responses to this relevant virus.

Ag2O Nanoparticles as a Candidate for Antimicrobial Compounds of the New Generation.

Antibiotic resistance in microorganisms is an important problem of modern medicine which can be solved by searching for antimicrobial preparations of the new generation. Nanoparticles (NPs) of metals and their oxides are the most promising candidates for the role of such preparations. In the last few years, the number of studies devoted to the antimicrobial properties of silver oxide NPs have been actively growing. Although the total number of such studies is still not very high, it is quickly increasing. Advantages of silver oxide NPs are the relative easiness of production, low cost, high antibacterial and antifungal activities and low cytotoxicity to eukaryotic cells. This review intends to provide readers with the latest information about the antimicrobial properties of silver oxide NPs: sensitive organisms, mechanisms of action on microorganisms and further prospects for improving the antimicrobial properties.

Impact of SARS-CoV-2 RBD Mutations on the Production of a Recombinant RBD Fusion Protein in Mammalian Cells.

SARS-CoV-2 receptor-binding domain (RBD) is a major target for the development of diagnostics, vaccines and therapeutics directed against COVID-19. Important efforts have been dedicated to the rapid and efficient production of recombinant RBD proteins for clinical and diagnostic applications. One of the main challenges is the ongoing emergence of SARS-CoV-2 variants that carry mutations within the RBD, resulting in the constant need to design and optimise the production of new recombinant protein variants. We describe here the impact of naturally occurring RBD mutations on the secretion of a recombinant Fc-tagged RBD protein expressed in HEK 293 cells. We show that mutation E484K of the B.1.351 variant interferes with the proper disulphide bond formation and folding of the recombinant protein, resulting in its retention into the endoplasmic reticulum (ER) and reduced protein secretion. Accumulation of the recombinant B.1.351 RBD-Fc fusion protein in the ER correlated with the upregulation of endogenous ER chaperones, suggestive of the unfolded protein response (UPR). Overexpression of the chaperone and protein disulphide isomerase PDIA2 further impaired protein secretion by altering disulphide bond formation and increasing ER retention. This work contributes to a better understanding of the challenges faced in producing mutant RBD proteins and can assist in the design of optimisation protocols.

From a recombinant key antigen to an accurate, affordable serological test: Lessons learnt from COVID-19 for future pandemics.

Serological tests detect antibodies generated by infection or vaccination, and are indispensable tools along different phases of a pandemic, from early monitoring of pathogen spread up to seroepidemiological studies supporting immunization policies. This work discusses the development of an accurate and affordable COVID-19 antibody test, from production of a recombinant protein antigen up to test validation and economic analysis. We first developed a cost-effective, scalable technology to produce SARS-COV-2 spike protein and then used this antigen to develop an enzyme-linked immunosorbent assay (ELISA). A receiver operator characteristic (ROC) analysis allowed optimizing the cut-off and confirmed the high accuracy of the test: 98.6% specificity and 95% sensitivity for 11+ days after symptoms onset. We further showed that dried blood spots collected by finger pricking on simple test strips could replace conventional plasma/serum samples. A cost estimate was performed and revealed a final retail price in the range of one US dollar, reflecting the low cost of the ELISA test platform and the elimination of the need for venous blood sampling and refrigerated sample handling in clinical laboratories. The presented workflow can be completed in 4 months from first antigen expression to final test validation. It can be applied to other pathogens and in future pandemics, facilitating reliable and affordable seroepidemiological surveillance also in remote areas and in low-income countries.

2021 Acknowledgment of mSphere Ad Hoc Reviewers

Yet, we accomplished it, in no small part due to years of fundamental research on how the immune system recognizes and responds to pathogens, how mRNAs are synthesized and translated, and how to effectively deliver macromolecules into cells. Much of this progress was anchored in the microbial sciences: studies of viral immunology, landmark work on mRNA metabolism in bacteria and virally infected cells, understanding how pathogens bind to and enter cells, development of techniques that were originally used to introduce viral oncogenes into mammalian cells in culture, and the use of viral vectors for gene therapy all set the stage for the COVID-19 vaccines. [...]we list below the many individuals who served as ad hoc reviewers in 2021. Kjersti Aagaard Mohamed M. H. Abdelbary Sabrina Absalon Michael C. Abt Mark D. Adams Josephine Azikuru Afema Kayode Olayinka Afolabi Surya D. Aggarwal Hector Aguilar-Carreno Christian Paul Ahearn Brian M. M. Ahmer Mustafa Akkoyunlu Md. Tauqeer Alam Ashraf Al Ashhab M. John Albert Anoop Alex Caroline Alfieri Holly M. Scott Algood Jonathan Allen Emma Allen-Vercoe Juan C. Alonso Francis Alonzo Christopher Alteri John Alverdy Christopher S. Anderson Matthew Zack Anderson David R. Andes Laura Maria Andrade De Oliveira Marco Andreolli Diego O. Andrey Alberto Antonelli Yoshiteru Aoi Cristian Apetrei Chelsie Elizabeth Armbruster Sandra K. Armstrong Jennifer M. Auchtung Tatjana Avšič-Županc Domenico Azarnia Tehran Sophie Bachellier-Bassi Michael A. Bachman Steffen Backert Matthew Baideme Camden R. Bair Jonathon L. Baker Katherine H. Baker Scott Baliban Jimmy D. Ballard Guilia Bandini Fernando Baquero Noa Barak-Gavish Joseph T. Barbieri Brianne Barker Jason C. Bartz Martine Bassilana Christine Marie Bassis Tilman Baumstark Marco Becherelli Sara Beier Daniel P. Beiting Georgios N. Belibasakis Aeriel D. Belk Samantha L. Bell Jessica A. Belser Jorge L. Benach Jose A. Bengoechea Peter Bergholz Teresa M. Bergholz Tanja Berić David Bernstein Stefan Bertilsson Ralph Bertram Sanchita Bhadra Dipankar Bhattacharyya Bijit Bhowmik Fadil A. Bidmos Claire H. Birkenheuer Jacob P. Bitoun Daniel Blanco-Melo Jon S. Blevins Joseph M. Bliss Patricia Pringle Bloom Antje Blumenthal Kasun H. Bodawatta Pierre Bogaerts Gregory Bonito Sébastien Bontemps-Gallo Angela Bordin Jens Bosse Anna Both Travis Bourret Kate Bowerman Eric Boyd Ethna Fidelma Boyd Todd Bradley Rita Branco Kyndall Braumuller Linda Breeden Mathieu Brochet Nichole A. Broderick Christopher B. Brooke Grayson Brown Jeremy S. Brown Kevin M. Brown Michael G. Brown Harry Brumer Donald A. Bryant Alison Buchan Lori L. Burrows Karen Bush Andrea Cabibbe Laty A. Cahoon Yi Cai Eloiza Helena Campana Edgar I. Campos-Madueno Eric Caragata Alessandra Carattoli Franck Gael Carbonero Miguel Carda Diéguez Jeffrey Carey Ryan B. Carnegie Jaime Carrasco Vern B. Carruthers Leslie S. Casey Irene Castano Santiago Castillo-Ramírez Clayton C. Caswell Rodrigo Cayô Daniel Cazares Brandi N. Celia Nuno Cerca Miguel Angel Cevallos Dipshikha Chakravortty Douglas L. Chalker Thomas M. Chambers Josephine R. Chandler Michael Chandler Robert L. Charlebois Sujata S. Chaudhari Neeraj Chauhan Damien Chaussabel Michael S. Chaussee Francisco P. Chavez Liang Chen Chiuping Cheng Rachel A. Cheng Laurent Roberto Chiarelli Alex W. H. Chin Michaelle Chojnacki Stephen A. Clark Erika C. Claud David W. Cleary Sara Clohisey Shira Milo Cochavi Darrell Cockburn Ashley Cohen Sean Conlan Laura Cook Gretchen Cooley Brendan Cormack Pierre Cornelis Caitlin Cossaboom Siobhan C. Cowley Robert A. Cramer Max Cravener Alison K. Criss Karissa L. Cross Robert W. Cross Liwang Cui Paul J. Cullen Natacha Cuoto Cameron R. Currie Todd Andrew Cutts Dennis G. Cvitkovitch F. Heath Damron Ajai A. Dandekar Stephen Daniels Biswadip Das Bryan W. Davies Charles R. Dean Jean-Winoc Decousser Elizabeth N. De Gaspari Miranda De Graaf Kirk W. Deitsch Harry P. De Koning Frank R. DeLeo Thomas G. Denes David W. Denning Rajendar Deora Cynthia Ann Derdeyn Steven C. Derrick Jigar V. Desai Lalitagauri Deshpande Sanjay Kumar Dey Vijaykrishn Dhanasekaran Rishu Dheer Robert P. Dickson Diego G. Diel Beatriz Diez Moreno Stephen P. Diggle Joseph P. Dillard Siyuan Ding Marc S. Dionne Alan Angelo Dispirito Dirk P. Dittmer Eunsoo Do Carlota Dobaño Lazaro Yohei Doi Janet Donaldson Caihong Dong Matthew J. Dorman Laurent Dortet Benoît Doublet Charles M. Dozois Jan Felix Drexler Yuchun Du Elves Duarte Edward G. Dudley Breck A. Duerkop Anne K. Dunn Sanjucta Dutta Kathryn Eaton Leo Eberl Kathryn M. Edenborough Tom Edlind Elizabeth A. Edwards Maren Eggers Sabine Ehrt Patrick Eichenberger Waldir P. Elias Jeremy R. Ellermeier Roland Elling Najib M. El-Sayed Mostafa S. Elshahed Joanne B. Emerson Virve Irene Enne Eeva Liisa Eronen-Rasimus Alice L. Erwin Javier Antonio Escobar-Perez Matthe J. Evans Franziska Faber Robert Fagan Christina S. Faherty Linda Falgenhauer Séamus Fanning Mauricio J. Farfan Matthew L. Faron Amy K. Feehan Mario F. Feldman Jinrong Feng J. Christopher Fenno David J. Ferguson Isabel Fernández Escapa Astrid Ferrer Richard L. Ferrero Kenneth A. Fields Joshua Fierer Sergio R. Filipe Maria F. Fillat Scott G. Filler Douglas K. Fischer Carlos Flores Stephanie Flowers Fabrizio Foieni Steven L. Foley Laura Ford Jarrod R. Fortwendel Michael T. France Kristi L. Frank Natalia Freund Georg Fritz Inga Fröding Takasuke Fukuhara Marta M. Gaglia Hannah Gaimster Raj Gaji James E. Galen Markus Ganter Michael G. Ganzle Erin C. Garcia Sarahi L. Garcia Amy Shirley Gargis Kathleen Gärtner Caroline Attardo Genco Noel Gerald Carmen Gherasim Lorenzo Giacani Heather L. Glasgow Oleg Glebov Erin S. Gloag Marek Gniadkowski Richard V. Goering Gustavo H. Goldman Jonathan Louis Golob Benjamin Golomb Laura Gómez-Consarnau Angela Gomez-Simmonds Yanhai Gong Jesus Gonzalo-Asensio Steven D. Goodman Tobias Goris Morgan Gorris Ria Goswami Matthias Gotte Revathi Govind Manish Goyal Andreas Grabrucker Lisa Gralinski Luke R. Green Alexander L. Greninger Finn Grey Elizabeth Grice Dennis Grogan Elisabeth Grohmann Trudy H. Grossman Cassandra Guarino Marc-Jan Gubbels Eric Guédon Pascale S. Guiton Arda Gulay Ravindra Kumar Gupta Gabriel Gutkind David Hackstadt Andrea Hahn Anders P. Hakansson Riley Hale Vanessa L. Hale Robert Hall Roy A. Hall Ruth M. Hall Brian K. Hammer Tobin Hammer Abdul N. Hamood Axel Georg Hamprecht Ken-Ichi Hanaki Lynn E. Hancock Blake M. Hanson Mingju Hao Md. Manjurul Haque Sohei Harada Clare Harding Lee H. Harrison Oliver Harschnitz Erica M. Hartmann Eric T. Harvill Asma Hatoum-Aslan Ben M. Hause Margo G. Haygood Cynthia Y. He Susu He Aoife T. Heaslip Nicholas S. Heaton Nagendra R. Hegde Christine Heilmann Henry S. Heine David E. Heinrichs Peera Hemarajata Tory A. Hendry Cristina Herencias Ana Hernandez Cordero Robert L. Hettich Andrés Hidalgo Steven Higgins Penelope Higgs Takahiro Hiono Itaru Hirai Theresa D. Ho Thomas Hoenen Nicole A. Hoff Deborah A. Hogan Peiying Hong Lauren Michelle Hook Thomas Hooven Alexander M. Horspool Paul A. Hoskisson Daniel K. Howe Gongzheng Hu Ke Hu Linden T. Hu Stephen S. H. Huang Eili Huhtamo Lewis Hun Jason F. Huntley Jillian H. Hurst Bonnie L. Hurwitz Wilhelmina Huston Justin Hutchison Albina Ibrayeva Melissa Ingala Thomas J. Inzana William W. Ja Mary Ann Jabra-Rizk Cody B. Jackson Anna C. Jacobs William R. Jacobs Guilhem Janbon Ingmar Janse Michael A. Jarvis Vicki Jeffers Niuniu Ji Dong-Yan Jin William Johnson Susan Joseph Lok R. Joshi Yuan Jun Barbara C. Kahl Maria Kalamvoki Suzanne R. Kalb Jeremy Phillip Kamil Manabu Kanno Fathi Karouia Anbu Kumar Karuppannan Fatah Kashanchi Joseph Keane Daniel B. Kearns Scott P. Keely Eliisa Kekäläinen Brendan Kelly Volkhard A. J. Kempf Arnaud Kengmo Tchoupa Nemat O. Keyhani Shabaana A. Khader Arifa