CRISPR Flashcards
What is the role of CRISPR in bacteria
It is an adaptive immune system, meaning that it uses past exposure to a pathogen to stimulate improved defense against future exposure to the same pathogen.
What are the steps of CRISPR
Spacer acuisition (invading DNA cleaved), crRNA biogenesis (CRISPR loci transcribed), target interference ( mature crRNAs associate with Cas and recruit them to invading phage DNA)
What is required for CRISPR
sgrna containing crRna and trcna sequences (involved in biogenesis), cas9
What are applications of CRISPR
reverse genetics by deleting a gene to learn its function. Mutating HNH and RUVc can create a dead version of Cas9 which binds but can’t cut target DNA. It can also produce GMOs.
What is gene drive -
a strategy to increasse the chances that a specific allele becomes more prevalent in a population
How were CRISPRs discovered
S thermophilus were exposed to a specific phage, and resistant bacteria possessed new spacers within their CRISPR loci with an exact sequence match to portions of the phage genome they had been exposed to
What do Cas genes do
They encode Cas proteins that function as DNases and RNases
What is true about Type II Cas9 protein
A single Cas9 protein is sufficient to mediate RNA guided viral DNA destruction during the interference step
What flanks the protospacer sequences selected by Cas9
Protospacer adjacent motif, required for Cas9 to cleave DNA
What are the two nuclease domains of Cas9 that cleave the DNA sequence
HNH domain that cleaves viral DNA complementary to crRNA
RuvC domain that cleaves the noncomplementary strand
What is the difference nonhomologous end joining and homology directed repair
NHEJ is a ligation of broken DNA fragments, is an error prone process and results in indels at the repair site
HDR uses an undamaged homologous chromosome or a sister chromatid as a template to correctly repair a broken chromosome allowing for complex substitutions, deletions, or additions. It is less error prone
What can you do with dead cas9
Genetically fuse the activation domain of a transcription activator to it in order to activate the transcription of any gene by designing an appropriate targeting sgrna
same thing can be done with repression, epigenetic tags, flourescent proteins, base conversions
What are off target effects of Crispr?
Crispr will make cuts at off target site in the genome, leading to a different sequence.
Where has crispr loci been identified
In 40 percent of bacteria species and 90 percent of archaea, another type of prokaryote
