Alvey (CRISPR) Flashcards
What does CRISPR stand for?
- clustered regularly interspersed short palindromic repeat array
How many recognition sites can Cas9 have, and how does this compare to normal REs?
- up to ≈20, compared to normal 6
What was CRISPR/Cas9 discovered as?
- bacterial immune system
How does bacterial adaptive immunity work?
- DIAG*
- CAS complex recognises foreign DNA and cleaves into small pieces
- incorp into genome at CRISPR loci
- transcribed and processed into mature crRNA
- crRNA and tracrRNA forms complex w/ Cas9
- crRNAs direct Cas9 complex to foreign DNA via 20bp seq homology adj to PAM site
- foreign DNA digested
What is the PAM site, and what is its role?
- protospacer adjacent motif
- determines specificity
- aspect changed in engineering, known as guide RNA
What stops Cas9 from cleaving its own DNA?
- doesn’t have a PAM site
What are the components of engineered CRISPR/Cas9 genome editing system?
- non-specific endonuclease (nearly always Cas9
- gRNA to direct nuclease to chosen site
Why are tracrRNA and crRNA joined together into single gRNA molecule for engineered CRISPR/Cas9 genome editing systems, and what else is is present?
- easier to transform 1 RNA into cell than 2
- inc presence of 20bp target seq and scaffolding seq to bind right Cas9 variant
How does Cas9 work?
- binds gRNA
- binds target DNA (as long as target is 5’ of PAM seq, or it can’t bind)
- cleaves target DNA to gen ds break
- break repaired by cell DNA repair machiner
What can cause a stem loop to form?
- palindromic seq
Why is the stem loop necessary in Cas9-gRNA complex?
- allows binding as recognition seq hangs out of complex
What 2 RNAs is native Cas9 targeted by and what are their roles?
- crRNA –> defines genomic target
- tracrRNA –> allows binding of crRNA and processing of pre-cRNA (chops up long pieces of RNA, not req in CRISPR gene editing as provide w/ already processed RNA
Why do target seqs need to be chosen which are adj to a PAM seq, and when can this be a problem?
- protects endogenous bacteria seq from being cut
- problem if no PAM seq in gene wnt to cut, but PAM variants give more choice
What is the role of PAM seqs?
- act as binding signal
What computer programme can be used to help find loci for CRISPR?
- CRISPR software matchmaker
What are the 2 methods of ds break DNA repair and when is each used?
- non-homologous end joining –> for gene KO
- homology directed repair –> for gene knock-in or replacement
How does repair by NHEJ work?
- DNA repaired in error prone manner by adding/removing bases so gene no longer functional
- happens when no template provided
How does repair by HDR work?
- DIAG*
- DNA template lines up next to break
- template invades gap and DNA synthesis gen complementary code
- when repair process complete, segment of template DNA inserted into break
- template separates, leaving original break w/ new section of code
What are the steps to designing CRISPR experiments?
1) known organism and seq
2) select gene to be manipulated
3) design gRNA to avoid off target activity
4) synthesise and clone gRNA
5) deliver Cas9 and gRNA (need vector and if hoping for HDR add template too)
6) validate genetic mod
What are some alts to CRISPR?
- zinc finger
- TALEN
- cre-lox
- recombineering
- sleeping beauty transposon
What is the aim of QTL analysis?
- find gene(s) responsible for known phenotype
How is QTL analysis carried out?
- chromosome region identified by freq co-occurrence of specific SNP w/ particular phenotype
- statistical process that seeks to identify regions of genome linked to QTLs
- mapping results in Lod scores and allows to identify region of chromosome assoc w/ quantitative trait of interest
- can’t find gene –> need further experimentation, eg. CRISPR
What is a real life eg. of CRISPR (beer)?
- QTL analysis to identify which genes important for prod desirable flavour in beer
- crossed 2 yeast strains (1 from ale and 1 from bioethanol plants)
- pooled segregates and measured 2-PEAc prod
- identified 2 genes responsible for 2-PEAc prod –> FAS2 and TOR1
- swapped high 2-PEAc alleles for low 2-PEAc alleles of FAS2 and TOR1, to see if these 2 genes alone could increase 2-PEAc prod –> it did!
Why is dCas9 used?
- provides versatile RNA-guided DNA targeting platform –> for reg and imaging genome, and rewriting epigenetic status
- all in seq-specific manner
What are the 2 forms of repurposed Cas9?
- nCas9 is a nickase (ss break)
- dCas9 is nuclease deactivated
How can Cas9 be repurposed?
- can be fused to other effectors to mediate site specific genetic and epigenetic reg w/o cleaving target DNA
How has Cas9 be repurposed for CRISPR-mediated epigenetic modifications, what is the assoc problem and how can this be approached?
- mod of epigenetic marks near dCas9 targeted site
- reg of downstream gene when targeted to enhancer/promoter
- epigenetic modifier makes chromatin more open/closed
- PROBLEM: introd ds breaks may create more problems than solves in whole organisms
- APPROACH: dev robust system for in vivo activation of endogenous target genes through trans-epigenetic remodelling
How was CRISPR-Cas9 used for epigenetic remodelling in vivo to treat mice models of type I diabetes?
- stimulated some liver cells to differentiate into pancreas-like cells, by increasing expression of Pdx1 gene
- resulted in increased insulin prod in disease model mice, via changing epigenome of diseased tissue and not changing gene seq
How are repression and activation of genes achieved by epigenetics?
- repression –> if H3K4me2 decreases, less chromatin and gene less likely to be expressed
- activation –> if H3K4me2 increases, gene turned on by HAT
How has CRISPR been used in research into Barth Syndrome?
- Barth Syndrome is mitochondrial cardiomyopathy
- showed caused by mutations in TAZ gene
- carried out CRISPR induced mutations in iPSCs from healthy donors
- to target TAZ used template w/ known Barth Syndrome mutation mutation in exon 6 of TAZ gene
- co-transfected w/ Cas9, treated w/ doxycycline on piggyBac transposon
- obtained Barth Syndrome mutants for further research
How can CRISPR be used for gene activation?
- CRISPRa
- fusion of p65 activation domain to dCas9 can activate reporter genes and endogenous genes w/ sgRNA to target specific promoter
- so can overexpress any gene
- recruits chromatin modifying complex
- DIAG*
What proteins have Cas9 variants (nCas9/dCas9) been used to direct to specific loci?
- deaminase (once repaired, replaces C w/ T base)
- transcriptional activators
- CRAB domain (transcrip repressor)
- fluorescent protein (eg. GFP)
