CRISPR Flashcards
What are the drawbacks of mouse models, and advantages does CRISPR have over genetic mice?
Lab strains are inbred which may harbour many background homozygous mutations
Also maintaiing stocks is expensive –> food, shelter, staff to feed them etc; also time consuming if waiting for animals eg age studies!
Not ideal for all human diseases –> eg mice and rats have different immune systems; primates might be better at demonstrating ToM
Ethical issues (Replacement: methhods which avoid use of animals; reduction \: minimiuse no. of animals used in experiement; refinement: minimise animal suffering, improve animal wellfare)
CRISR is less time consuming and more efficient, because you can directly target the GoI (using cas9 and gRNA) and entry efficiancy is much higher (vs mice where genetic recombination is a rare event)
also you bypass the chicmeric stage; you inject trans gene directly into the blastocyst (rather than harvesting the mutant gene from ES cells) so 100% of offspring will have germline transmission of transgene!
Compare Forward vs Reverse Genertics
In forward genetics you know the phenotype (eg small bee vs queen bee), so you do genotyping (eg nanopore) to trace the locus of that phenotype.
In reverse genetics you do a kenetic KO or KI to observe what effects this has on phenotype. You might use CRISPR, then check for off-target effects using nanopore and SS.
What genetic KD methods preceded CRISPR (clue KD not KO!)
RNA inteference using small intefering RNA (siRNA).
This causes KD of a gene due to incomplete depletion of a protein –> so some may remain thus KD is only temporary.
- relies on small intefering RNA
- this is good for crude experiments eg to see if a gene is active or not, for example BDNF in synaptic scaling.
- there are offtarget effects
- shRNA plasmid DNA plus gene silencers inderted into cell.
shRNA is transcribed so that it incorporates the gene silenceing region. Then this is processes by dicer into siRNA –> siRNA is activated by RISC (RNA interactive silencing complex) –> cleaves at target mRNA sites
-another example is role of ENSE and ARPP19; when these proteins are KD by siRNA you don’t see segregation during anaphase (live cell imaging) –> use control siRNA test on western blot that the addition of RNA is causing any changes to phenotype alone
What is the purpose of Large scale siRNA screens?
You incorporate GFP protein into the promotor region for the gene you are targetting, then reverse transfect the wells on the plate that have siRNAs inside. This will intefere with transcription, so GFP will be turned off in KD/KI cells.
Crude (things might not be KD enough to see a fluoresence effect) but also quick and easy
Why is genetic editing with plasmid vectors inefficient in human cells?
- very rare event –> for every billion cells transfected you only get about 100 hits
- also homologous arms need to be >4kb long !
so overall gene targetting really hard to do in human cells
Draw and label the CRISPR-Cas9 complex
How does CRISPR work?
Bacteriophages insert their viral DNA into cells; CRISPR can cut some of the viral genome and store it as their own ‘CRISPR array’ –> this acts like an imulogical memory of different infections.
The CRISPR array genome gets transcribed into precrRNA –> this couples with Cas9
CRISPR-Cas9 complex searches the cell for the same sequences as the guide RNA –> acts as viral defence by making a double strand break in the target DNA.
So how can we haness CRISPRs mechanisms and use it for genetic KO or KI?
You want to induce NHEJ (non homologous end joining) for KO, because deletions will affect the sequence of AA thus leading to non-fuctional protein ~(due to a frame-shift in the bases). For KI you want a targeted insertion to change one of the bases to swap it to another AA, changing the function of the protein.
First step is to design the short guide-RNA. This is the region of DNA CRISPR-Cas9 will taget, ie the specific gene you want to intefere with. You want ~20bp target specific crRNA, must be next to PAM site (NGG) - note gRNA doesnt contain a PAM site itself, but it must be next to one. Uniqueness is important here to reduce off taregt effects. You can design multiple guides eg exons 2,3,4,5 to ensure the whole gene is KO.
Then you do the gene editing. You inject your CRISPR-Cas9 RNP into pool of ESCs. The Cas9 will cause DSBs at the target DNA site (3-4 nucleotides upstream of PAM site) the cell will undergo DSB repair by either NHEJ or HDR.
NHEJ will result in single insertion or deletion which will cause a frameshift in the bases so disrupt the order of AA –> leading to a non-functional protein.
HDR will insert and new KI sequence (you can manipulate the system by cleaving the proteins that regulate NHEJ, or by injecting CRISPR-Cas9 during S/G2 phase of cell cycle); you can inject these KI sequences along with the CRISPR-Cas9 RNP (ssODNs).
Then you have to analyze the cells to see which ones have been genetically edited.
What kind of DSB repair do you want for KO and KI, respectively?
For KD you want NHEJ because you can induce indels –> leading to non functional proteins as frame shift of bases changes AA sequence. You could also knock in a gene with this since one base substitution could change the AA sequence, but it would be better to do DSB repear by HR because then you can incorporate a new DNA sequence
What are the different modes of CRISPR-Cas9 delivery? Why is delivery difficult and what method is preffered?
- standard plasmid transformation with DNA: clone gRNA and Cas9 into a plasmid vector
- alterantively deliver the RNA with Cas9 directly into cells
- Or , if using eggs/embryos –> directly inject Ribonucleoprotein (Cas9 complex with gRNA) (RNP) this is favoured method as it has the least off-target effects and works quickly!
Delivery can be difficult because not all cells are amenable to transfection
Other methods incluse microfluidics –L> uses microscoping channels to manipulate fluids and delicer the CRISPR-Cas9 cargo into cells –> involves making cells transiently permeable when pushing through an array on microfluidics chip, so the small CRISPR-Cas9 molecule can pass through the membrane –> cand deliver CRISPR cargo to millions of cells in seconds, but it is still a develoing method so limitations are unknown; there is some difficulty with chips becoming clogged with cells
How might you assess KO/KI efficiency?
- Indel Screening: loss of resriction site
If your target DNA is near a restriction site, you can test for this as doing PCR probably won’t reveal single indels. Therefore, you treat your PCR products with a restriction enzyme, and then you separate using gel electrophoresis. The shorter fragments (that have been cleaved by restriction enzyme) will travel further, thus you know these ones have been edited. Problem is this is labourious and you have to clone the genetically edited cells first. Further, it is limited by the presenceof a restriction site! - SS InDel screening using ICE (online program) which tells you which proportion of your cells have been sucessfully edited –> although this doesnt really tell you if your protein is functional or not, because InDels can still give rise to functional proteins. Uses SS to tell you the probability of the Indel or KI sequence too. Much more efficent method
Why are KIs harder to do than KOs? How can you optimise KI efficiency?
default DSB repair in humans is NHEJ –> HR only happens at specific points in the cell cycle
- synchronise cells at S/G2 phase (like they did in the CRISPR paper)
- chemically inhibit SCR7 protein –> this inhibits NHEJ
How can you identify off-target effects of CRISPR-Cas9?
- in vitro –> illumina based digenome-seq. Here, genomic DNA is digested in vitro by nucleases, this yields multiple DNA fragments with identical 5’ or 3’ ends at on or off target sites. Then the DNA is subjected to WGS (eg illumina), giving sequence reads with identical 5’ ends corresponding to DSBs. Then with a computer progam you align the 5’ ends so that sequence reads are aligned vertcally at cleavage sites. –> this method capture off-target dsites with an indel frequency of 0.1% or lower. Tells you where DSBs have occured.
- in vivo –> Guide-seq from illumina. DSO (double-stranded oligodeoxynucleotides) are incorporated into the DSB - this is a reporter oligo and will locate where DBs have occured in live human cells
- silico prediction algorithms - biased
in what ways can CRISPR knock down a gene?
- tissue specific promotors
- temporal specific (ie during a specific developmental window)
- inducible eg drug tamoxifen activated by oestrogen receptor which carries Cas9 into the nucleus
how might CRISPR be used in cancer tretament?
It could knock in a stop codon upstream of an oncogene
