Crispr Flashcards
CRISPR acronym
Clustered Regulalry Interspaced Palindromic Repeats
what is CRISPR
It is the hallmakr of the bacterial defence system and is part of their adaptive immune system
-It is used by researcehrs in modifying genes permanently in living cells and organsisms
-Allows for the correction of mutations at precise locations to treat genetic causes of diseases
SHERLOCK
Uses CRISPR-Cas13 to target RNA and is used as a sensitive diagnostic tool
Innate Immunity
An interna system that can be used to defend against foregin pathogens
-restricitons enzymes are involved in this
-RE coding sequences are fixed andd protein structure remains stable for long periods of time
Adaptive immunity
A form of immunity that can change or adapt when exposed to new/different pathogens
-Eg. Vaccines
CRISPR background
-Discovered when investigating bacterial immunity
-CRISPR is a form of adaptive immunity
-Now been manipulated for genetics and molecular biology research
CRISPR discovery
CRISPR loci has been found in 40% of bacteria and 90% of arcahae
CRSIPR spacer sequences
identical to fragments of bacteriophage genomes
-Viral sequences within CRISPR loci serve as molecular memory of previous viral attacks
Experimental evidence of CRISPR in adaptive immune system
-Exposed S.thermophillus to specific phage
-Bacterial cells that survived became resistant to that phage but not others
-Resistant bacteria possessed new spacers within their CRISPR loci with an exact sequence match to portions of the phage genome they were exposed to
-Deletion of new spacer abolished phage resistance
-Experimental insertions of new viral sequence-derived spacers into the CRISPR loci of sensitive bacteria rendered them resistant.
CRISPR-Cas mechanism principle
-RNA guided destruction of invading DNA (nucleotuides)
-Adaptive immunity is also dependant on a set of adjacent CRISPR-associated (cas) genes
-Cas genes encode Cas proteins that function as DNases and RNases
CRISPR-cas mechanism steps
-Spacer acquisition
-crRNA biogenesis
-Target interference
Spacer acquisition
-Invading phage DNA is cleaved into smaller fragments known as protospacers, which are then inserted into CRISPR loci to become new spacers
-When new spacers are added to the CRISPR locus, repeat sequences are duplicated such that each spacer is flanked by repeats in each side
crRNA biogenesis
CRISPR loci are transcribed starting att the promoter within the leader, into long RNA transcripts known as pre-crRNA
-Processed into short CRISPR-derived RNAs (crRNAs), each containing a single spacer flanked on both sides by repeat sequences
Target Interference
-Mature crRNAs associated with Cas nucleases, or nuclease complexes, and recruit them to complementary sequences in invading phage DNA
-Cas nucleases then cleave the viral DNA, thus neutralising infection
Type I and Type 3 CRISPR-Cas systems
Type 1- Cas3 and Type III- Cas10
-Require multi-subunit protein complexes to mediate RNA-guided viral DNA destruction during the interference step