EDICIÓN GENÉTICA CRISPRcas
EDICIÓN GENÉTICA CRISPRcas
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repair
template
+donor DNA
CRISPRs
Cleavage
Guide RNA
Cas9 Protein
Nuclease-based genome editing
33-34 amino acid sequence – only 12th and 13th residue vary – and determine
nucleotide binding.
We can construct these arrays, add a nuclease and use for genome editing
Fok1
TGAGGAGGCGGCAACGGCGGGCGCCGGGGCGGCGGGCCCCGGGGCGAGCA
ACTCCTCCGCCGTTGCCGCCCGCGGCCCCGCCGCCCGGGGCCCCGCTCGT
Fok1
Cleavage
Zinc Finger Nucleases (ZFN)
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CRISPR-cas
RECONOCER
ANA
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Identification of CRISPR
Spacer
CRISPR
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SISTEMA CRISPR/CAS COMO MECANISMO DE DEFENSA
http://www.addgene.org/CRISPR/guide/
CRISPR/cas requires:
– the Cas9 protein
– an engineered small guide RNA (sgRNA) with a PAM
sequence upstream of target complementary sequence
– A designed hairpin to mimic the CRISPR RNA and trans-
acting RNA complex.
– Base-pairing between the sgRNA and target DNA causes
double-strand breaks due to the endonuclease activity of
Cas9
http://www.addgene.org/CRISPR/guide/
Qi et al. Cell 2013
DNA target does not need to be unique and can appear in
multiple locations, all of which will be targeted by the Cas9
nuclease for cleavage.
The sgRNA can bind on either strand of DNA and the Cas9
will cleave both strands (double strand break, DSB). The
DSB results in the silencing of that DNA sequence.
La naturaleza palindrómica
Repeteciones palindrómicas
: FASE DE TRANSCRIPCIÓN Y MADURACIÓN DE crRNA
Transcripción del loci de CRISPR- cas para generar un complejo guía de RNA-
proteína.
Se transcribe el locus de CRISPR y el tracrRNA. Luego Cas9 une el tracrRNA con el
transcrito primario e inmaduro de CRISPR.
Este es cortado por una RNAasa III quedando de forma madura como crRNAs. El
dúo maduro de crRNA:tracrRNA permanence unido a Cas9 como un hetero-
duplex. .
FASE DE INTERFERENCIA
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CRISPR BLAST
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RNAi-like CRISPR-Cas system (CASS)
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Programming CRISPR
Complementary to:
RNA and coding strand of DNA
Template strand
First case of directly programming CRISPR-based immunity—a flu shot for bacteria.
Self-splicing intron
Plasmid Plasmid
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Discovery of tracrRNA
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Flexibility in CRISPR processing systems
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Each Cas9 nuclease domain cleaves one DNA strand
Cas9 uses two nuclease domains to cleave the two strands in the target DNA
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Cleavage sites in target DNA PAM, protospacer adjacent motif
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Secondary structure of Dual-tracrRNA: crRNA
S. pyogenes
L. innocua
N. meningitidis
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Crystal Structure of Cas9/gRNA Complex and Target DNA
I: Genome modification
Monkey Rice
Liang, P., et al., 2015, Protein Cell, 6(5): 363-372. (Received March 30, 2015 Accepted April 1, 2015)
Cpf1, a Single RNA-Guided Endonuclease