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Transcript of CRISPR/Cas9 Prezi
Found naturally in archaea and bacteria
Repetitions of DNA sequences
Spacer DNA sequences between the repeats
Typically found before CRISPR repeat-spacer regions
As many as 45 are thought to exist
CRISPR/Cas systems are used in the adaptie immune system of 90% of archaea and 40% of bacteria. They remember and destroy foreign DNA from sources such as bacteriophages (viruses that infect bacteria.)
CRISPR is not the first nuclease-based gene editing technique; others include the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs).
In the lab...
(Clustered Regularly Interspaced Short Palindromic Repeats)
An endonuclease (enzyme that cuts DNA)
The Cas9 complex consists of:
A guide RNA (gRNA) sequence that tells Cas9 where to cut the DNA
A short sequence of foreign DNA (protospacer) is inserted in front of the CRISPR sequence
When the DNA is transcribed the resulting RNA is known as precursor CRISPR RNA (pre-crRNA)
The pre-crRNA are bound to trans-acting CRISPR RNA (tracrRNA)
The pre-crRNA/tracrRNA duplexes are cut by endoribonucleases (enzymes that cut RNA) into individual crRNA/tracrRNA duplexes
These duplexes are referred to as the guide RNA (gRNA)
The gRNA is combined with Cas9
When Cas9 finds a foreign DNA sequence that is complimentary to the gRNA, Cas9 endonuclease cleaves the DNA strand.
The foreign DNA sequence targeted by Cas9 must have a 3-6 nucleotide sequence known as a protospacer adjacent motif (PAM) at the beginning to be recognized and cut. This prevents the integrated DNA from being cleaved.
Cas9 knows where to stop cleaving DNA based on a counting mechanism, so the sequence targeted in research is always 20 nucleotides (DNA bases) long.
A DNA sequence in the gene of interest is inserted into the gRNA of another organism (e.g. a bacteria)
A Cas9 plasmid vector is created that will be introduced to the target organism
(Irreversible) Gene Knockdown
Seeks to irreparably stop the function of the target gene
Typically done to organisms in very early development (before cells have begun differentiating)
Reversible Gene Knockdown: CRISPR interference (CRISPRi)
Seeks to block transcription of DNA into RNA
Uses catalytically dead Cas9 (dCas9) that can't cut DNA
Targets the promoter region of a gene
Activates inactive genes, allowing the transcription of DNA.
Uses the same technique as CRISPRi, but a transcriptional activator is attached to the dCas9
Creation of model organisms
Learning about the functions of specific genes
The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats
Grissa, et al., 2007. doi: 10.1186/1471-2105-8-172
CRISPR-Based Adaptive Immune Systems
Terns, et al., 2011. doi: 10.1016/j.mib.2011.03.005
CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
Barrangou et al., 2007. doi: 10.1126/science.1138140
Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs.
Hale, et al., 2012.
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.
Jinek, et al., 2012. doi: 10.1126/science.1225829
Highly improved gene targeting by germline-specific Cas9 expression in
Kondo, et al., 2013. doi: 101534/genetics,113.15673
DNA interrogation by the RNA-guided endonuclease Cas9
Sternburg, et al., 2014. doi: 10.1038/nature13011
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression
Qi, et al., 2013. doi:10.1016/j.cell.2013.02.022
CRISPR/Cas9 image: http://www.genecopoeia.com/product/crispr-cas9/
CRISPR sequence image: http://wwwnc.cdc.gov/eid/article/16/4/09-1435-f3
CRISPRs were discovered in 1987 and named in 2000, but their potential for gene editing was not proven until 2013.