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CRISPR Nick Turner Bio 446 Fall 16 REVIEW • siRNA • dsRNA • microRNA • Immunity • Gene Regulation • Enzymatic breakdown of RNA RNA INTERFERENCE • RNAi • The use of RNA to inhibit gene expression. • Guiding RISC (RNA Induced Silencing Complex) cleave and degrade specific segments of RNA DISCOVERY OF CRISPR • Ishino et al, 1987 iap (isozyme conversion of alkaline phosphates) genes in E. Coli Observed repeats near the 3’ end of the gene of ~29bp repeats and spaced by ~32bp “spacers” The conserved region is palindromic DISCOVERY OF CRISPR • Mojica et al, 2000 The Short Regularly Spaced Repeats (SRSRs) was present in many species. Proposed that it either had an important function, or was an ancestral byproduct. However, The spacer between each repeat varied between organisms. • Jansen et al, 2002 Four Cas genes near the repeats with helicase, and nuclease protein domains. • Together coined the term CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats describing the loci FUNCTION OF CRISPRS • Bolotin et al, Pourcel et al, and Mojica et al 2005 Independently determined spacer DNA are identical to viral and some plasmid DNA. FUNCTION OF CRISPR • Barrangou et al, 2007 Experimentally supported CRISPR immunity hypothesis. Streptococcus Thermophilus 100% match between the spacers and bacteriophages provided resistance Anywhere between1 to 15 nucleotide polymorphisms would not provide immunity. FUNCTION OF CRISPR • Barrangou et al, 2007 • Editing, modifying, and deleting various segments of WT phi858 +S1S2 CRISPR ASSOCIATED SYSTEMS • CAS Enzymes • Bolotin et al, 2005 • Helicase, Nuclease, DNA integration, Exonuclease • CAS5 vs CAS9 CRISPR ASSOCIATED SYSTEMS • Bhaya et al 2011 PROTOSPACER ADJACENT MOTIF • PAM • A part of the target DNA, but not the CRISPR locus. • Allows Cas9 to bind to foreign DNA • Protects the cell DNA from being fragmented • 5’- NGG -3’ MOLECULAR MECHANISM • Sapranauskas et al 2011 Engineered a protospacer & PAM motif onto plasmids Plasmids were successfully cleaved Mutations of PAM stop the CRISPR mechanism Confirming Cas9 nuclease activity • Deltcheva et al 2011 The palindromic repeat is used to guide and bind to Cas9 • Jinek et al, 2012 Cas9 creates a double stranded cleave CRISPR vs RNAi CRISPR TARGETS DNA • Marraffini and Sontheimer 2008 • Garneau et all 2010 Transcribed CRISPR RNA binds to DNA S. Thermophilus plasmids Southern Blotting CRISPR/CAS9 SYSTEM • Charpentier and Doudna S. Pyogenes crRNA, tracrRNA, and gRNA • Cong et al 2013 The CRISPR/CAS9 system functions in eukaryotic cells. Human, and mouse • Gene Insertion OTHER GENOME EDITING TECHNIQUES • TALEN/ Zinc Finger Nucleases APPLICATIONS • Research • Medicine Providing a better understanding of genes that are not well understood yet. Flies, Zebra Fish, Mice, Cells Single gene diseases. Huntington's, Neurofibromatosis, Phenylketonuria, Deafness: Tang et al, 2016. Gene insertion Multiple gene disease: Parkinsons: Soldner et al, 2016 Designer Babies REFERENCES • • • • • Ishino, Yoshizumi, et al. "Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product." Journal of bacteriology 169.12 (1987): 5429-5433. Mojica, Francisco JM, et al. "Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria." Molecular microbiology 36.1 (2000): 244-246. Mojica, Francisco JM, Jesús García-Martínez, and Elena Soria. "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements." Journal of molecular evolution 60.2 (2005): 174-182. Bolotin, Alexander, et al. "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin." Microbiology 151.8 (2005): 2551-2561. Pourcel, C., G. Salvignol, and Gilles Vergnaud. "CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies." Microbiology 151.3 (2005): 653-663. • Brouns, Stan JJ, et al. "Small CRISPR RNAs guide antiviral defense in prokaryotes." Science 321.5891 (2008): 960-964. • Sapranauskas, Rimantas, et al. "The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli." Nucleic acids research (2011): gkr606. • Deltcheva, Elitza, et al. "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471.7340 (2011): 602-607. • Jinek, Martin, et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096 (2012): 816-821. • Soldner, Frank, et al. "Parkinson-associated risk variant in distal enhancer of αsynuclein modulates target gene expression." Nature (2016). • Tang, Zi-Hua, et al. "Genetic Correction of Induced Pluripotent Stem Cells From a Deaf Patient With MYO7A Mutation Results in Morphologic and Functional Recovery of the Derived Hair Cell-Like Cells." Stem Cells Translational Medicine 5.5 (2016): 561-571. • Heidenreich, Matthias, and Feng Zhang. "Applications of CRISPR-Cas systems in neuroscience." Nature Reviews Neuroscience 17.1 (2016): 36-44. • Jansen, Ruud, et al. "Identification of genes that are associated with DNA repeats in prokaryotes." Molecular microbiology 43.6 (2002): 1565-1575. • Sontheimer, Erik J., and Luciano A. Marraffini. "Microbiology: slicer for DNA." Nature 468.7320 (2010): 45-46. • Bhaya, Devaki, Michelle Davison, and Rodolphe Barrangou. "CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation." Annual review of genetics 45 (2011): 273-297. • Garneau, Josiane E., et al. "The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA." Nature 468.7320 (2010): 67-71. • Barrangou, Rodolphe, et al. "CRISPR provides acquired resistance against viruses in prokaryotes." Science 315.5819 (2007): 1709-1712.