Gene Therapy Flashcards
Discuss the principle gene therapy using lentiviruses to treat neurodegenerative diseases in a symptomatic or disease-modifying manner
When was CRISPR technology first published?
2012 by Jinek et al
What is CRISPR?
Clustered regulatory interspersed short palindromic repeats - unique DNA sequence segments transcribed into RNA identified in bacterial DNA in 1987
What is Cas9?
A RNA-DNA binding nuclease
Describe CRISPR-Cas adaptive immunity
During CRISPR administration, the injection of phage DNA into bacterial cells activates the Cas1-Cas2 adaptation proteins. These excise spacer-sized fragments of phage DNA and incorporate it into the CRISPR array, which is transcribed into RNA and cleaved, then processed at repeats to generate crRNAs. Individual crRNAS are found by Cas9. When a phage with DNA matching a CRISPR spacer enters, Cas9 finds and pairs the crRNA and DNA, destroying the phage
Describe the method of action of Cas9
Cas9 binds to tracer RNA, which recruits crRNA via homologous sequences. Cas9 loaded with both RNAs scans DNA for PAM sequences, then binds to PAM and opens its adjacent double helix to determine homology. When it finds homology, it becomes a nuclease and cleaves both strands of the RNA close to the PAM
Where is the PAM found?
In the phage DNA spacer - but not in the CRISPR array containing the spacers, allowing Cas9 to distinguish bacterial genome from invader phage DNA and only cleave RNA
How does interaction of Cas9 with RNA and DNA alter its function?
Cas binding to PAM changes its conformation (allosteric change) to make it a DNA denaturing enzyme. When it finds complete homology with crRNA, it becomes a DNA nuclease
How much homology with crRNA is required to activate Cas9 nuclease activity?
20 nucleotides
How many mitmatches with crRNA will prevent Cas9 cleavage?
1-2 are sufficient
Name 2 methods which mammalian chromosomes use to repair DNA after Cas9 cleavage
Non-homologous end-joining, homologous directed recombination
State a way in which Cas9 proteins can be modified
Mutated to become single-strand nucleases, nuclease activity deactivated (dCas9)
State a use of nuclease-deactivated Cas9
Fusing dCas9 to GFP allows fluorescent marking of DNA
Name some methods of delivery of CRISPR-Cas9
Microinjection, transfection, viral vectors, gold particles
Name some viral vectors
Lentiviruses, adenoviruses, adeno-associated viruses
State 3 current challenges around the therapeutic applications of gene editing
Delivery, controlling repair pathways, ethics
