The CRISPR revolution and its potential impact on global health security

References

• Medicine, N. A. of S., Engineering, and, Division, H. and M., Health, B. on G. & States, C. on G. H. and the F. of the U. Global health and the future role of the United States. (2017) doi:https://doi.org/10.17226/24737.
   Google Scholar
• Carroll D. Genome editing: past, present, and future. Yale J Biol Med. 2017;90(4):653–659.
   Google Scholar
• Knott GJ, Doudna JA. CRISPR-Cas guides the future of genetic engineering. Science. 2018;361(6405):866–869.
   Google Scholar
• Mehand MS, Al-Shorbaji F, Millett P, et al. The WHO R&D Blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts. Antiviral Res. 2018;159:63–67.
   Google Scholar
• Whitehouse CA. Crimean-Congo hemorrhagic fever. Antiviral Res. 2004;64(3):145–160.
   Google Scholar
• WHO Ebola Response Team. After ebola in West Africa–unpredictable risks, preventable epidemics. N Engl J Med. 2016;375:587–596.
   Google Scholar
• Smith GJD, Vijaykrishna D, Bahl J, et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. 2009;459(7250):1122–1125.
   Google Scholar
• SteelFisher GK, Blendon RJ, Bekheit MM, et al. The public’s response to the 2009 H1N1 influenza pandemic. N Engl J Med. 2010;362(22):e65.
   Google Scholar
• Chen H, Smith GJD, Zhang SY, et al. Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature. 2005;436(7048):191–192.
   Google Scholar
• World Health Organization. Zika virus, microcephaly and Guillain-Barré syndrome situation report. (2016).
   Google Scholar
• Spížek J, Novotná J, Řezanka T, et al. Do we need new antibiotics? The search for new targets and new compounds. J Ind Microbiol Biotechnol. 2010;37(12):1241–1248.
   Google Scholar
• Herfst S, Schrauwen EJA, Linster M, et al. Airborne transmission of influenza A/H5N1 virus between ferrets. Science. 2012;336(6088):1534–1541.
   Google Scholar
• Imai M, Watanabe T, Hatta M, et al. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature. 2012;486(7403):420–428.
   Google Scholar
• Jr WCW. Legionnaires disease: historical perspective. Clin Microbiol Rev. 1988;1(1):60–81.
   Google Scholar
• Zhao Z, Zhang F, Xu M, et al. Description and clinical treatment of an early outbreak of severe acute respiratory syndrome (SARS) in Guangzhou, PR China. J Med Microbiol. 2003;52(8):715–720.
   Google Scholar
• Zaki AM, Boheemen S, Van, Bestebroer T, et al. A. D. M. E. & Fouchier, R. A. M. isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012;367(19):1814–1820.
   Google Scholar
• Burki T. Outbreak of coronavirus disease 2019. Lancet Infect Dis. 2020;20(3):292–293.
   Google Scholar
• Goba A, Khan SH, Fonnie M, et al. An outbreak of ebola virus disease in the lassa fever zone. J Infect Dis. 2016;214(suppl 3):S110–S121.
   Google Scholar
• Smith WAndrews CH, Laidlaw PP, et al. Obtained from influenza patients. Lancet. 1933;222(5732):66–68.
   Google Scholar
• Samji T. Influenza A: understanding the viral life cycle. Yale J Biol Med. 2009;82:153–159.
   Google Scholar
• Barik S. New treatments for influenza. BMC Med. 2012;10(1):104.
   Google Scholar
• Hussain M, Galvin HD, Haw TY, et al. Drug resistance in influenza A virus: the epidemiology and management. Infect Drug Resist. 2017;10:121–134.
   Google Scholar
• Zost SJ Parkhouse K, Gumina ME et al. Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains. Proc. Natl. Acad. Sci. U.S.A. 114, 12578–12583 (2017).
   Google Scholar
• Sanjana NE, Cong L, Zhou Y, et al. A transcription activator-like effector toolbox for genome engineering. Nat Protoc. 2012;7(1):171–192.
   Google Scholar
• Urnov FD, Rebar EJ, Holmes MC, et al. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010;11(9):636–646.
   Google Scholar
• Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32(4):347–355.
   Google Scholar
• Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–821.
   Google Scholar
• Tsang J, LaManna CM. Open sharing during COVID-19: CRISPR-based detection tools. Crispr J. 2020;3(3):142–145.
   Google Scholar
• Martin-Laffon J, Kuntz M, Ricroch AE. Worldwide CRISPR patent landscape shows strong geographical biases. Nat Biotechnol. 2019;37(6):613–620.
   Google Scholar
• Brinegar K, Yetisen AK, Choi S, et al. The commercialization of genome-editing technologies. Crit Rev Biotechnol. 2017;37(7):1–12.
   Google Scholar
• Erp PB, Van, Bloomer G, Wilkinson R, et al. The history and market impact of CRISPR RNA-guided nucleases. Curr Opin Virol. 2015;12:85–90.
   Google Scholar
• Murray J. The class of 2019: five synthetic biology companies to watch this year. synbiobeta. https://synbiobeta.com/the-class-of-2019-five-synthetic-biology-companies-to-watch-this-year/ (2019). Web. 15 Dec. 2020.
   Google Scholar
• Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. U.S.A. 109, E2579–86 (2012).
   Google Scholar
• Jiang F, Doudna JA. CRISPR-cas9 structures and mechanisms. Annu Rev Biophys. 2017;46(1):505–529.
   Google Scholar
• Jiang F, Zhou K, Ma L, et al. A Cas9-guide RNA complex preorganized for target DNA recognition. Science. 2015;348(6242):1477–1481.
   Google Scholar
• Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759–771.
   Google Scholar
• Chen JS, Ma E, Harrington LB, et al. CRISPR-Cas12a target binding unleashes single-stranded DNase activity. Science. 2017;360:1–29.
   Google Scholar
• Swarts DC, Oost J van der, Jinek M. Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a. Mol Cell. 2017;66(2):221–233.e4.
   Google Scholar
• Yan WX, Hunnewell P, Alfonse LE, et al. Functionally diverse type V CRISPR-Cas systems. Science. 2018;363:eaav7271–9.
   Google Scholar
• East-Seletsky A, O'Connell MR, Knight SC, et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature. 2016;538(7624):270–273.
   Google Scholar
• Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016;353(6299):aaf5573–aaf5573.
   Google Scholar
• Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438–442.
   Google Scholar
• Smargon AA, Cox DBT, Pyzocha NK, et al. Cas13b is a type VI-B CRISPR-associated RNA-guided rnase differentially regulated by accessory proteins Csx27 and Csx28. Mol Cell. 2017;65(4):618–630.e7.
   Google Scholar
• Yan WX, Chong S, Zhang H, et al. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein. Mol Cell. 2018;327-339.e5.
   Google Scholar
• Harrington LB, Burstein D, Chen JS, et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science. 2018;362(6416):839–842.
   Google Scholar
• Karvelis T, Bigelyte G, Young JK, et al. PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Res. 2020;48(9):5016–5023.
   Google Scholar
• Larson MH, Gilbert LA, Wang X, et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc. 2013;8(11):2180–2196.
   Google Scholar
• Chen B, Gilbert LA, Cimini BA, et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell. 2013;155(7):1479–1491.
   Google Scholar
• Oakes BL, Nadler DC, Flamholz A, et al. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nature. 2015;34:646–651.
   Google Scholar
• Polstein LR, Gersbach CA. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat Chem Biol. 2015;11(3):198–200.
   Google Scholar
• Amabile A, Migliaria A, Capasso P, et al. Inheritable silencing of endogenous genes by hit- and-run targeted epigenetic editing. Cell. 2016;167(1):219–224.e14.
   Google Scholar
• Zaboikin M, Zaboikina T, Freter C, et al. Non-homologous end joining and homology directed dna repair frequency of double-stranded breaks introduced by genome editing reagents. PLoS ONE. 2017;12(1):e0169931.
   Google Scholar
• Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533(7603):420–424.
   Google Scholar
• Hess GT, Tycko J, Yao D, et al. Methods and Applications of CRISPR-mediated base editing in eukaryotic genomes. Mol Cell. 2017;68(1):26–43.
   Google Scholar
• Komor AC, Zhao KT, Packer MS, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv. 2017;3(8):eaao4774.
   Google Scholar
• Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. 2017;551(7681):464–471.
   Google Scholar
• Li X, Wang Y, Liu Y, et al. Base editing with a Cpf1–cytidine deaminase fusion. Nat Biotechnol. 2018;36(4):324–327.
   Google Scholar
• Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA editing with CRISPR-Cas13. Science. 2017;550:eaaq0180–15.
   Google Scholar
• Vogel P, Moschref M, Li Q, et al. Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs. Nat Methods. 2018;15(7):535–538.
   Google Scholar
• Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149–157.
   Google Scholar
• Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154(2):442–451.
   Google Scholar
• Mandegar MA, Huebsch N, Frolov EB, et al. CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell. 2016;18(4):541–553.
   Google Scholar
• Abudayyeh OO, Gootenberg JS, Essletzbichler P, et al. RNA targeting with CRISPR–Cas13. Nature. 2017;550(7675):280–284.
   Google Scholar
• Konermann S, Lotfy P, Brideau NJ, et al. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell. 2018;173(3):665–676.e14.
   Google Scholar
• Strutt SC, Torrez RM, Kaya E, et al. RNA-dependent RNA targeting by CRISPR-Cas9. eLife. 2018;7:e32724.
   Google Scholar
• Dugar G, Leenay RT, Eisenbart SK, et al. CRISPR RNA-dependent binding and cleavage of endogenous RNAs by the campylobacter jejuni Cas9. Mol Cell. 2018;69(5):893–905.e7.
   Google Scholar
• Price AA, Sampson TR, Ratner HK, et al. Cas9-mediated targeting of viral RNA in eukaryotic cells. Proc. Natl. Acad. Sci. U.S.A. 112, 6164–6169 (2015).
   Google Scholar
• Smalley E. CRISPR mouse model boom, rat model renaissance. Nat Biotechnol. 2016;34(9):893–894.
   Google Scholar
• Ma D, Liu F. Genome editing and its applications in model organisms. Genomics Proteomics Bioinformatics. 2015;13(6):336–344.
   Google Scholar
• Sun S-H, Chen Q, Gu HJ, et al. A mouse model of SARS-CoV-2 infection and pathogenesis. Cell Host Microbe. 2020;28(1):124–133.e4.
   Google Scholar
• Zotova A, Zotov I, Filatov A, et al. Determining antigen specificity of a monoclonal antibody using genome-scale CRISPR-Cas9 knockout library. J Immunol Methods. 2016;439:8–14.
   Google Scholar
• Thompson NC, Zyonts S. Who tries (and who succeeds) in staying at the forefront of Science. Social Science Research Network. 2017:1-52.
   Google Scholar
• Parnas O, Jovanovic M, Eisenhaure TM, et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell. 2015;162(3):675–686.
   Google Scholar
• Mohr SE, Perrimon N. RNAi screening: new approaches, understandings, and organisms. Wiley Interdiscip Rev RNA. 2012;3(2):145–158.
   Google Scholar
• Fellmann C, Gowen BG, Lin P-C, et al. Cornerstones of CRISPR–Cas in drug discovery and therapy. Nat Rev Drug Discov. 2016;16(2):89–100.
   Google Scholar
• Wang R, Simoneau CR, Kulsuptrakul J, et al. Genetic screens identify host factors for SARS-CoV-2 and common cold coronaviruses. Cell. 2020;184(1):106-119.e14.
   Google Scholar
• Schneider WM, Luna JM, Hoffmann HH, et al. Genome-scale identification of SARS-CoV-2 and pan-coronavirus host factor networks. Cell. 2020;184(1):120-132.e14.
   Google Scholar
• Wei J, Alfajaro MM, DeWeirdt PC, et al. Genome-wide CRISPR screens reveal host factors critical for SARS-CoV-2 infection. Cell. 2020184(1):76-91.e13.
   Google Scholar
• Lobato IM, O’Sullivan CK. Recombinase polymerase amplification: basics, applications and recent advances. Trends in analytical chemistry: TRAC. 2018;98:19–35.
   Google Scholar
• Ackerman CM, Myhrvold C, Thakku SG et al. Massively multiplexed nucleic acid detection using Cas13. Nature. 2020;582(7811):277-282.
   Google Scholar
• Pardee K, Green AA, Takahashi MK, et al. Rapid, low-cost detection of zika virus using programmable biomolecular components. Cell. 2016;165(5):1255–1266.
   Google Scholar
• Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018;360(6387):444–448.
   Google Scholar
• Gootenberg JS, Abudayyeh OO, Kellner MJ, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018;360(6387):439–444.
   Google Scholar
• Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. Medrxiv Prepr Serv Heal Sci 2020.05.04.20091231 (2020) doi:https://doi.org/10.1101/2020.05.04.20091231.
   Google Scholar
• Broughton JP, Deng X, Yu G, et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870–874.
   Google Scholar
• Fozouni P, Son S, de León Derby MD, et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell. 2020;184(2):323-333.e9.
   Google Scholar
• Li H, Yang Y, Hong W, et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther. 2020;5:1–23.
   Google Scholar
• Lee K, Conboy M, Park HM, et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng. 2017;1(11):889–901.
   Google Scholar
• Yang W, Tu Z, Sun Q, et al. CRISPR/Cas9: implications for modeling and therapy of neurodegenerative diseases. Front Mol Neurosci. 2016;9:30.
   Google Scholar
• Yin C, Zhang T, Qu X, et al. In vivo excision of HIV-1 provirus by SaCas9 and multiplex single-guide RNAs in animal models. Mol Ther. 2017;25(5):1168–1186.
   Google Scholar
• The SM. The CRISPR/Cas9 system: their delivery,in vivo and ex vivo applications and clinical development by startups. Biotechnol Prog. 2017;33(4):1035–1045.
   Google Scholar
• Pancino G, Saez-Cirion A, Scott-Algara D, et al. Natural resistance to HIV infection: lessons learned from HIV‐exposed uninfected individuals. J Infect Dis. 2010;202:345–350.
   Google Scholar
• Soppe JA, Lebbink RJ. Antiviral goes viral: harnessing CRISPR/Cas9 to Combat Viruses in Humans. Trends Microbiol. 2017;25(10):833–850.
   Google Scholar
• White MK, Hu W, The KK. CRISPR/Cas9 genome editing methodology as a weapon against human viruses. Discov Med. 2015;19(105):255–262.
   Google Scholar
• Abbott TR, Dhamdhere G, Liu Y, et al. Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and Influenza. Cell. 2020;181(4):865–876.e12.
   Google Scholar
• Gholizadeh P, Kose S, Dao S, et al. How CRISPR-Cas system could be used to combat antimicrobial resistance. Infect Drug Resist. 2020;13:1111–1121.
   Google Scholar
• Bikard D, Euler CD, Jiang W, et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol. 2014;32(11):1146–1150.
   Google Scholar
• Kang YK, Kwon K, Ryu JS, et al. Nonviral genome editing based on a polymer-derivatized crispr nanocomplex for targeting bacterial pathogens and antibiotic resistance. Bioconjugate Chem. 2017;28(4):957–967.
   Google Scholar
• Gomaa AA, Klumpe HE, Luo ML, et al. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio. 2014;5(1):e00928–13.
   Google Scholar
• Citorik RJ, Mimee M, Lu TK. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol. 2014;32(11):1141–1145.
   Google Scholar
• Hammond A, Galizi R, Kyrou K, et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature. 2016;34:78–83.
   Google Scholar
• Slomovic S, Pardee K, Collins JJ Synthetic biology devices for in vitro and in vivo diagnostics. Proc. Natl. Acad. Sci. U.S.A. 112, 14429–14435 (2015).
   Google Scholar
• Schmid-Burgk JL, Gao L, Li D, et al. Highly parallel profiling of Cas9 variant specificity. Mol Cell. 2020;78(4):794–800.e8.
   Google Scholar
• Tsai SQ, Zheng Z, Nguyen NT, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nature. 2015;33:187–197.
   Google Scholar
• Tsai SQ, Nguyen NT, Malagon-Lopez J, et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat Methods. 2017;14(6):607–614.
   Google Scholar
• Akcakaya P, Bobbin ML, Guo JA, et al. In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature. 2018;2:914.
   Google Scholar
• Kim D, Bae S, Park J, et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nature. 2015;12(3):237–243
   Google Scholar
• Chen JS, Dagdas YS, Kleinstiver BP, et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature. 2017;550(7676):407–410.
   Google Scholar
• Kleinstiver BP, Pattanayak V, Prew MS, et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529(7587):490–495.
   Google Scholar
• Wang Z, Pan Q, Gendron P, et al. CRISPR/Cas9-derived mutations both inhibit HIV-1 replication and accelerate viral escape. Cell Rep. 2016;15(3):481–489.
   Google Scholar
• Makarova KS, Zhang F, Koonin EV. SnapShot: class 2 CRISPR-Cas Systems. Cell. 2017;168(1–2):328.e1.
   Google Scholar
• Shmakov S, Smargon A, Scott D, et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nature Rev Microbiol. 2017;15(3):169–182.
   Google Scholar
• Bak RO, Porteus MH. CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep. 2017;20(3):750–756.
   Google Scholar
• Charlesworth CT, Deshpande PS, Dever DP, et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans. Nat Med. 2019;25:249–254.
   Google Scholar
• Kim S, Koo T, Jee H-G, et al. CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 2018;28(3):367–373.
   Google Scholar
• Kirkpatrick J, Koblentz GD, Palmer MJ, etl. Editing biosecurity: needs and strategies for governing genome editing. (2018).
   Google Scholar
• Cyranoski D CRISPR-baby scientist fails to satisfy critics. 13–14 (2018).
   Google Scholar
• Wang H, Li J, Li W, et al. CRISPR twins: a condemnation from Chinese academic societies. Nature. 2018;564(7736):345.
   Google Scholar
• Normile D Organizers of gene-editing meeting blast Chinese study but call for ‘pathway’ to human trials. (2018).
   Google Scholar
• Law, C. on S., Technology, and, Affairs, P. and G., Medicine, N. A. of S., Engineering, and & Olson, S. International summit on human gene editing: a global discussion. (2016) doi:https://doi.org/10.17226/21913.
   Google Scholar
• Lander ES, Baylis F, Zhang F, et al. Adopt a moratorium on heritable genome editing. Nature. 2019;567(7747):165–168.
   Google Scholar
• Gronvall GK. The scientific response to COVID-19 and lessons for security. Survival. 2020;62(3):77–92.
   Google Scholar
• Burwell SM, Townsend FF, Bollyky TJ, et al. Improving pandemic preparedness: lessons from COVID-19. https://www.cfr.org/report/pandemic-preparedness-lessons-COVID-19/pdf/TFR_Pandemic_Preparedness.pdf (2020). Web. 15 Dec. 2020.
   Google Scholar
• Board GPM A world in disorder. https://apps.who.int/gpmb/assets/annual_report/GPMB_AR_2020_EN.pdf; (2020). Web. 15 Dec. 2020.
   Google Scholar
• Biodefense, B. C. on. Diagnostics for biodefense: flying blind with no plan to land. https://biodefensecommission.org/wp-content/uploads/2020/11/Diagnostics-Special-Focus_final_web2-1.pdf (2020). Web. 15 Dec. 2020.
   Google Scholar