ZENG CUME Flashcards
What is CRISPR
CRISPR (/ˈkrɪspər/) (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea.[2] These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form of acquired immunity.[2][3][4][5]
*NOTE the sequences are between the palindromic repeats - the spacers are the foreign dNA
What is a PAM
A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.[1] The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence.[2][3][4][5] PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.
How many CRIPSR types/classes are there
2 classes and 6 types
defined evolutionarily
2 classes are class -mutliple effector protein, class1 - only one protein
What are some common CRISPR diagnosis methods
Guide Directed reconsistution of split proteins by catalytically inactive cas 9 partners
Cas 9 based desctruction of PAM containing sites
Cas 9 causing unwinding of non targeted DNA to then isothermally amplify
NASBACC – (Nucleic acid sequence based amplification – CRISPR CLEAVAGE) (see figure)
Combines 3 things – amplification, PAM dependant detection and a toehold
So 1st NASBA amplifies RNA fragment through reverse transcription
If theres a PAM sequence – we cleave it
If not – we generate the whole RNA sequence (Full length RNA) –
This activates the TOEHOLD switch (causes a color change)
LEOPARD – leveraging engineered tracrRNA and on target DNA for parallel RNA detection
Key things we need we need for next gen diagnostics–
single nucleotide specificity but also ease of use and cost efficiency (uses isothermal amp)
WHat are the 3 major types in class 2
TYPE II - cas 9 - cis cleavage
TYPE V - CAS 12 - trans cleavage targets DNA
TYPE VI - trans cleavage targets RNA
HOW DOES SHERLOCK WORK?
Sherlock is TYPE VI uses Cas13 - So essentially you have your sample and you replicate the target sequence to amplify - and you add in reporter
THEN you add in Case 13 wth cRNA for the target - which activates CAS 13 which starts trans cleavage of anything nearby - it gets the reporters (which are quencher +fluorophore and creates signal
-can be set on a lateral flow assay where uncleaved reporter quencher is bound at one line and cleaved binds at a different line
CRISPR generation
so the bacetriophage DNA gets incorporated in as spacer between plaindromic repeats - this is transcibed into pre cRNA
There is also tracrRNA - COMPLEMENTARY to the palindromic repeats - so can hybridize with -NOTE - only cas 9 uses TRACR no case 12 or cas 13
so 3 things - pre cRNA , tracrRNA and cas9
RNASE III cleaves to make cRNA complexes that defend and this is the Cas complex typically thought of
It goes and tries to cut things recognized to cRNA - IT RECOGNIZES VIRAL GENOME vs its own BAsed on the PAM
The key difference between crRNA tracrRNA and gRNA is that crRNA is one of the two types of RNA of CRISPR, which is complementary to the target DNA sequence, while tracrRNA is the second type of RNA of CRISPR, which serves as a binding scaffold for the Cas nuclear
when we do CRISPR - we combine the Tracr and cRNA as sgRNA - and this with CAS is a functional complex
What are some example of CRIPSR DIAGNOSIS methods
guide directed reconsistution of split proteins by acatycally inactive cas 0 partners
Cas 9 based destruction of PAM containing sites - classic
Cas 9 unwinding of DNA to amplify
Explain Nasbacc
So it works on the workflow on ZIKA RNA - the default workflow is that we amplify the RNA with NUCLEIC ACID SEQUENCE BASED AMPLIFICATION (NASBA) - this activates a TOEHOLD sensor - and different things can happen - can either create a signal or in this case it opened d a ribosome binding site to allow for translation of lac Z which would create a colorimetric readout. THe cas plays a role in that if it recognizes this amplified RNA will cut it and truncate it - essentially ruining this workflow
IN the paper it was the same thing except RNA was amplified/converted to ds DNA and this ds DNA binds the toehodler trigger
Sherlock vs DETECTR
sherlock cleaves RNA reporters DETECTR cleaves DNA reporters - similarly at the beginning for replication - SHERLOCK uses RPA while DETECTR uses RT RPA (reverse transcription - because needs to convert to RNA first from DNA
LOD ranges from amp vs no amp
picmolar vs atto or zeptomolar
What are some issues with amplification and what are some workaround
ampligicaiotn often might lose specific info like epigenetics - we can try to increase the signal without amplification - one strategy is using an additional nuclease in addition to our Cas (this is with Cas 13) - Lwacas13a - cleaves differently - cleaves uridine which allows for our other RNA nuclease to cleave adenosine rich sequences which increases the signal
Can you do quant with cripsr and what types?
Yes can do rel and absolute
absolute requires external cal curve
- the signal here relates to collateral cleavage and cant be combined with amplification (in one pot)
pico-mM range
NEED TO make sure if amplifying not saturating
What is CARMEN
Combinatorial arrayed reactions for multiplexed evaluation of nucleic acids
OK so you have an array and in each you have two droplets – one is your amplified nucleic acid target sample (so I guess going down the row maybe different amplified targets)
The other is a LwaCas13 detection mix – each for a specific target and solution based colour code In fluorous oil
They record once initially to understand what each cell is and then apply an external electric field to merge the droplets
Once merged can tell which ones fluoresce
*Im confused as to what they’re amplifying here
Maybe can look at the paper
PROS of this method: not time consuming spotting – higher scalability
Fast high throughput low cost – buit requires preamplification, microscopy analysis and skilled lab personnel.
Reduces reagent cost as well
Read paper
