What are the three tools for studying genes and proteins?
1. cloning and site-directed mutagenesis
2. protein expression in a variety of systems
3. purification of proteins for analysis
What is the major limitation of studying genes and proteins?
you have removed the gene/protein from its natural environment
what is CRISPR/Cas9?
A bacterial immune system that can be exploited to edit genes in all organisms
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
What are cas genes?
CRISPR-associated genes that are highly conserved and typically adjacent
What are the spacers in CRISPR?
viral gene sequences
What is CRISPR?
repeat sequences found in >40% of sequenced bacterial genomes, the spacers are viral sequences
What are the steps of interaction in CRISPR as a bacterial adaptive immune system?
1. Cas gene is transcribed into Cas protein and the viral sequences with spacers (CRISPR) is transcribed into pre-crRNA
2. the pre-crRNA is processed into crRNA
3. the crRNA interacts with tracrRNA
4. tracrRNA interacts with cas9, helping load the crRNAs
How do tracrRNAs mediate interaction with Cas?
they are an invariable sequence that interact with the "constant" region of the crRNAs, mediating the loading of the crRNAs into the Cas protein
How does CRISPR act as a bacterial adaptive immune system?
1. the viral DNA base-pairs with crRNA loaded into Cas9
2. Cas9 cleaves the viral DNA, destroying it
3. When a new virus is encountered, other Cas proteins process the viral genome and add the sequence to the CRISPR array (this is the adaptive part) so th
What are the two parts of the system for targeted genome editing (CRISPR)?
Cas9 and single guide RNA (sgRNA)
What are the benefits of CRISP/Cas9?
-it is easy to use and therefore is accessible to any laboratory with simple molecular biology tools
-it is highly programmable and can be directed to any location on the genome
-it is highly multiplexable which allows for many genomic sites to be targete
What happens to the (viral) DNA after it's cleaved?
there is non-homologous end-joining (NHEJ) during which endogenous DNA repair machinery attempts to fix the break; generally this introduces mutations that destroy the gene
-repair template DNA must also be provided