CRISPR Flashcards
3 Types of CRISPR
Acquisition: a portion of foreign DNA is inserted into the CRISPR array
Expression: the CRISPR array is transcribed and cleaved to form many small crRNAs
Interference: the crRNAs containg homologies to infecting agents. If the crRNA/Cas complex recognizes an infectious (foreign) DNA or RNA, it will bind to it and target it for degradation
CRISPR
technology that creates double stranded breaks at precis location in a genome. The host’s repair mechanisms fix the break
Emmanuelle Charpentier
Works on CRISPR system where only 1 CAs protein that binds to the crRNAs.
DISCOVERY: A second RNA (tracrRNA) was also required. when Cas9/crRNA/tractRNA were together, the complex would find viral DNA sequences and get rid of them.
Gene therapy
Addition of a correct copy of the gene into cells in the target organ or tissue
Genome editing
alteration fo the genome at a specific location to alter the genetic sequence
Goal of gene therapy and genome editing
to introduce Dna to correct the function of an abnormal gene
RNAi
RNA interference
Cell have evolved RNAi for gene regulation. Adapted RNAi to control gene expression in the lab (“knock-downs”)
Jennifer Doudna and Emmanuelle Charpentier
Cas9/crRNA/tracr complex made a double stranded, blunt end, cut in the viral DNA at precise location near a short sequence (NGG) called PAM. The cleaved viral RNA is then targeted for degradation
Realized if they replcaed the viral sequence with any sequence, they could create a double stranded break at a specific site anywhere in the genome
Create a plasmid with _____. introduce plasmid into a cell. let the cell do the rest of the work:
Cas9 and guide RNA
Transcribe guide RNA, transcribe and translate CAS9, cleave genome at precise location and repair cleavage using NHEJ or HR
Off-target effects
Unintended mutations at untargeted genomic sites that occur during genome edit
