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This review explores the bacterial innate immune system, focusing on the CRISPR-Cas9 technology and its applications in genome editing and disease treatment. The review also discusses the limitations of CRISPR and provides relevant references.
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C.R.I.S.P.R. James Matt Johnson
Bacterial Innate Immune System (a review..) • Restriction enzymes • Enzymes with endonuclease activity that cut at very specific sequences. • Sequences are hard-wired • Extracellular substances to block bacteriophage • Substances on surface of cell that block infection • Abortive Infection • Bacteria cell self-destructs to prevent phage growth and spread
So…. what is CRISPR? • Clustered Regularly Interspaced Short Palindromic Repeats • Commonly referred to as CRISPR-Cas9 • Ultimately, CRISPR is a way for bacteria to adapt and defend itself against bacteriophage • Analogous to human adaptive immunity (ONLY KIND OF!!)
Design of CRISPR-Cas9 system Key Terms: • Protospacer adjacent motif (PAM) sequence • tracrRNA • crRNA • Cas http://symposcium.com/2014/03/qa-what-is-crispr-technology/
How does CRISPR work within bacteria? Step 1: Adaptation https://www.researchgate.net/publication/319010479_Interdependencies_Between_the_Adaptation_and_Interference_Modules_Guide_Efficient_CRISPR-Cas_Immunity
How does CRISPR work within bacteria? Step 2: RNA Biogenesis https://www.researchgate.net/publication/319010479_Interdependencies_Between_the_Adaptation_and_Interference_Modules_Guide_Efficient_CRISPR-Cas_Immunity
How does CRISPR work within bacteria? Step 3: Interference https://www.researchgate.net/publication/319010479_Interdependencies_Between_the_Adaptation_and_Interference_Modules_Guide_Efficient_CRISPR-Cas_Immunity
Applications Vector-born Illnesses
Applications cont’d… Hereditary Illnesses
Applications cont’d… Other Diseases/Illness
Limitations of CRISPR-Cas9 • Human immune system • Embryonic editing and expression events • Gene sequence specificity issues • PAM Sequence requisite
References • Abedon, Stephen T. “Bacterial ‘Immunity’ against Bacteriophages.” Bacteriophage, 1 Jan. 2012, pp. 50–54., www.ncbi.nlm.nih.gov/pmc/articles/PMC3357385/. • Biolabs, New England. “CRISPR/Cas9 & Targeted Genome Editing: New Era in Molecular Biology.” New England Biolabs: Reagents for the Life Sciences Industry, www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology. • “CRISPR Timeline.” Broad Institute, 20 Mar. 2018, www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline. • Crudele, Julie M., and Jeffrey S. Chamberlain. “Cas9 Immunity Creates Challenges for CRISPR Gene Editing Therapies.” Nature Communications, vol. 9, no. 1, 2018, doi:10.1038/s41467-018-05843-9. • DelViscio, Jeffery, and Dominic Smith. “How CRISPR Works, Explained in Two Minutes.” STAT, STAT, 3 Apr. 2018, www.statnews.com/2018/04/04/how-crispr-works-visualized/. • Fernández, Clara Rodríguez. “Seven Diseases That CRISPR Technology Could Cure.” Labiotech.eu, Labiotech UG, 22 Aug. 2018, labiotech.eu/tops/crispr-technology-cure-disease/. • Flora, Adriano, and Jochen Welcker. “CRISPR Genome Engineering: Advantages and Limitations.” Rodent Research Models, 22 Feb. 2017, www.taconic.com/taconic-insights/gems-design/crispr-genome-engineering-advantages-limitations.html. • “How Bacterial Cell Recognizes Its Own DNA.” Phys.org - News and Articles on Science and Technology, Phys.org, phys.org/news/2015-04-bacterial-cell-dna.html. • Jurberg, Arnon D., and Paul J. Brindley. “Gene Function in Schistosomes: Recent Advances toward a Cure.” Frontiers in Genetics, vol. 6, 2015, doi:10.3389/fgene.2015.00144. • Ka, Donghyun, et al. “Molecular Organization of the Type II-A CRISPR Adaptation Module and Its Interaction with Cas9 via Csn2.” Nucleic Acids Research, Aug. 2018, doi:10.1093/nar/gky702. • Knight, Matty, et al. “Schistosomes and Snails: a Molecular Encounter.” Frontiers in Genetics, vol. 5, 2014, doi:10.3389/fgene.2014.00230. • Lim, Youngbin, et al. “Structural Roles of Guide RNAs in the Nuclease Activity of Cas9 Endonuclease.” Nature Communications, vol. 7, Feb. 2016, p. 13350., doi:10.1038/ncomms13350. • Nuñez, James K, et al. “Cas1–Cas2 Complex Formation Mediates Spacer Acquisition during CRISPR–Cas Adaptive Immunity.” Nature Structural & Molecular Biology, vol. 21, no. 6, Apr. 2014, pp. 528–534., doi:10.1038/nsmb.2820. • Public Affairs, and UC Berkeley. “How CRISPR Works.” Berkeley News, 15 Feb. 2017, news.berkeley.edu/2017/02/15/how-crispr-works-and-what-it-can-do/. • Qi, Lei. “Faculty of 1000 Evaluation for Diversity and Evolution of Class 2 CRISPR-Cas Systems.” F1000 - Post-Publication Peer Review of the Biomedical Literature, 2017, doi:10.3410/f.727234466.793532246. • Semenova, Ekaterina, and Konstantin Severinov. “Interdependencies Between the Adaptation and Interference Modules Guide Efficient CRISPR-Cas Immunity.” Evolutionary Biology: Self/Nonself Evolution, Species and Complex Traits Evolution, Methods and Concepts, 2017, pp. 51–62., doi:10.1007/978-3-319-61569-1_3. • Xiao, Yibei, et al. “How Type II CRISPR–Cas Establish Immunity through Cas1–Cas2-Mediated Spacer Integration.” Nature, vol. 550, no. 7674, Apr. 2017, pp. 137–141., doi:10.1038/nature24020.