Transcript crispr - UNM Biology

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
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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.
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Brouns, Stan JJ, et al. "Small CRISPR RNAs guide antiviral defense in prokaryotes."
Science 321.5891 (2008): 960-964.
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Sapranauskas, Rimantas, et al. "The Streptococcus thermophilus CRISPR/Cas system
provides immunity in Escherichia coli." Nucleic acids research (2011): gkr606.
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Deltcheva, Elitza, et al. "CRISPR RNA maturation by trans-encoded small RNA and
host factor RNase III." Nature 471.7340 (2011): 602-607.
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Jinek, Martin, et al. "A programmable dual-RNA–guided DNA endonuclease in
adaptive bacterial immunity." Science 337.6096 (2012): 816-821.
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Soldner, Frank, et al. "Parkinson-associated risk variant in distal enhancer of αsynuclein modulates target gene expression." Nature (2016).
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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.
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Heidenreich, Matthias, and Feng Zhang. "Applications of CRISPR-Cas systems in
neuroscience." Nature Reviews Neuroscience 17.1 (2016): 36-44.
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Jansen, Ruud, et al. "Identification of genes that are associated with DNA
repeats in prokaryotes." Molecular microbiology 43.6 (2002): 1565-1575.
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Sontheimer, Erik J., and Luciano A. Marraffini. "Microbiology: slicer for DNA." Nature
468.7320 (2010): 45-46.
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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.
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Garneau, Josiane E., et al. "The CRISPR/Cas bacterial immune system cleaves
bacteriophage and plasmid DNA." Nature 468.7320 (2010): 67-71.
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Barrangou, Rodolphe, et al. "CRISPR provides acquired resistance against
viruses in prokaryotes." Science 315.5819 (2007): 1709-1712.