Lecture #5 - CRISPR Part 1 Flashcards
Editting Technology Before CRISPR
Before CRISPR - had Talens
Talens = Fuse a protein that binds DNA and a protein that cuts DNA
- Sequence specificity is hardwired int the proteins (protein only binds to that sequence for the lifetime of that protein)
- Makes sequence specific cuts
Bacteriophages
Bacteriophages = major threat to bacteria
- Injects genetic material into the bacteria cell
When the Bacteriophages injects genetic material into the bacteria the viral genetic material is preferentially transcribed and translated
Original Purpose of CRISPR
CRISPR/cas9 = evolved as a bacterial adaptive immune system to target the specific sequences of invading bacteriophage DNA and kill foreign DNA (Ex. Kill Phages or plasmids)
- Adaptive immune (has memory) - Adaptive because CRIPSR remembers previous encounters with phages/plasmids
How does CRIPSR work in bacterial immunity
Upon infection by a novel pathogen CRSIPR cas identifies viral threat and integrates part of the viral DNA into the bacterial genomes at the CRISPR locus –> the sequence in the CRISPR locus is transcribed and paired with cas nuclease –> programs the cas to specifcally cut the bacterial genome –> THEN upon reinfection by the same virus strain –> CRISPR cas9 can identify and neutralize the viral threat
- Progaming of cas = through base complementaryity using the crRNA
What happens during infection by bacteriophage
During infection by bacteriophage fragments of viral DNA can be acquired into a genome CRIPSR array –> allows for “genetic memory of infection”
Memory in CRISPR = Spacers
CRISPR (Overall)
CRIPSR – Cluster Regularly interspersed palindromic Repeats
Overall - Processed crRNA from CRISPR locus complexes with a cas nuclease to cleave viral DNA in a sequence-specific manner —> prevents future infections
- crRNA – CRISPR RNA
CRISPR/Cas9 System overall (key points)
- 20 BP target RNA is fused with 76 RNA scafoloed
- NGG PAM sequence -
- PAM = protospacer adjacent motif (ex. Cas looks for NGG)
- PAM is needed because then Cas9 would cut the bacterial CRISPR array memory in the genome itself
- PAM is NOT in the bacteria CRISPR memory but IS in the phage that you are targeting - Creates a dsDNA break 3 BP upstream from the PAM
- Breaks repaired by error prone NHEJ or HDR
Image - blue is DNA target ; green is gRNA
Where does gRNA come from in CRISPR
gRNA = comes from the Spacer DNA in CRISPR –> make gRNA –> cas9 look for DNA that matches the guide
gRNA ALSO comes form tracrRNA
- tracrRNA = acts as a scafold
Bacteria need tracerRNA and CRISPR crRNA VS. In lab = fuse crRNa and the tracerRNA
What does Cas9 look for
Cas9 = looks for DNA that matches the spacer AND looks for PAM sequence
- Cas9 matches the spacer and DNA BUT next to that match there needs to be a PAM (NGG for Cas9)
Cas9 Nuclease domains
cas9 = gas Two nuclease domain
RubC and HnH = 2 nuclease domains on cas9 –> each cleaves 1 starnd of target = get dsDNA break
Cas = Ccntinues to cut until a mutation is introduced
Cells repairing the dsDNA break
Repair = where editing actually starts
The cell can repair the break to the original sequence –> in this case there is NO editting
Cells can repair the dsDNA break using:
1. NEHJ = sticks dsDNA back together
- Often results in INDELS at the cut site –> NOW have edited DNA because repaired the DNA wrong
- Once have INDELS = cas9 can’t cus the sequence again because it no longer matches the guide (prevents cas9 form cutting DNA again)
2. HDR –> Have a dsDNA breal in 1 sister chromatid–> use the sister chromatic on the other chromosome as a template to corect the broekn sequence
- IF you overwhelm the cells with a template that has the edit that you want THEN when you cut the DNA it will repair the DNA using the donor DNA instead of the sister chromatid = can insert the sequence that you wanted to add to th cell
Cas9 binding to the DNA
R loop = DNA that match the memory is unwound when cas9 binds to DNA
- 1 srand is bound to the guide and one strand is free ssDNA
What do cas genes code for
Upstream of the array = cas proteins themselves
- Cas protein = involoved in new sequence (spacer) integration + CRISPR RNA processing + Interference
CRISPR locus
CRISPR locus = array with unique spacers targeting discrete viral sequences
- CRSIPR locus = has a library of guides that target many unique viral sequences
Locus alternats a constant repeat sequence and virus-specific spacer sequences
- On each side of the space = has short repeats (Spacer (virus specific) –> Repeat –> Spacer –> Repeat etc.)
Spacer
Spacer = short DNA sequence from the Phage (20-23 BP of DNA stole from the phage that is integrated into the CRIPSR array and becomes memory)
- IF have a new infection by a pahge that matches the spacer (matches the 30 BP of memory) –> THEN the phage will be destroyed by the CRIPSR system
- Bacteria can make new memories
- Spacer = gives the specificity for each virus/memory
- makes CRISP adaptive
Purpose - acts as memory because the crRNA is loaded onto cas9 and cuts anything that matches
Arrays can contain tens to hundreds of spacers
crRNA
Pre-crRNA are transcribed from an upstream leader seqeucne then processed into single mature crRNAs
- Upstream leader seqeunce = upstream of the spacer and repeat sequences (red in the image)
- Pre-crRNA transcript matures into multiple shorter segments (shorter segments that each target motifs)
crRNA = contains the 20-nucleatide spacer sequence used for base-pairing with target DNA
- From the integrated viral sequence at the CRISPR locus (from the Spacers)
- crRNA = variable
Cas9 Nuclease complex
Cas9 complex = programmed to cut specific DNA sequence by interrogating for PAM sequence THEN base pairing to the spacer sequence of the crRNA
Cas9 complex = composed of cas9 portein and crRNA + tracrRNA
Cas9 = has 2 nuclease domains = can make dsDNA break in the target DNA
- Cuts are only made IF the DNA sequence properly matches the spacer sequence that is encoded in the crRNA
- cas9 = binds to DNA bases using an RNA guide
tracrRNA
tracrRNA = strcutual element that tethers the crRNA (complex the crRNA and the cas9 protein)
If the protospacer and spacer sequences stored in the bacterial genome are the same –> THEN how does the CRISPR cas9 system differentiate between foreign viral DNA and its won genome at the CRISPR array locus
Cas protein recognzies a PAM sequence downstream of the spacer/portospacer base pairing on the dsDNA
PAM
PAM (protosoacer Adjacent Motif) - a several nucleotide seqeunce
PAM = needed for Cas to dock on the DNA and open up the DNA THEN can test the crRNA for complementarity and have cleavage
- CRISPR array does NOT have PAM –> NOT encoded in the crRNA = cas will not cut the CRISPR array in the bacterial genome
Purpose - Seraching for PAMs first allows teh cas9 complex to avoid self-taregting and allows cas9 to survey the DNA more efficiently
Classes of CRISPR systsems
CRISPR systems exist in two main classes (based on how many proteins bind to the associated RNA)
Class 1 – Several proteins complex to form nuclease
- Multiple proteins bind to the RNA to become active
- Has Types 1,3, and 4 systems
Class 2 – Single nuclease effector (One protein binds RNA)
- Includes Types 2, 5, and 6 systems
Other CRIPSR systems – Types 3 = transcription dependent ; Type 1 chew up DNA in a unpredictable manner
Why use Type 2 CISRP systems in the lab
Type 2 = used for gene editing in lab because it is simply (only requires 1 cas protein to bind to crRNA)
Type 2 ALSO makes precise dsDNA cuts which triggers the host DNA damage response
SpCas9 in Lab
Scietitists use Streptococcus Pyogenes Cas9 (spCas9) in the lab for genome edittig
How do we adapt the bacterial immune system to cut DNA in Eukryotic cells:
1. A nuclear localization signal is added to enable nuclear import in Eukaryotes (brings the cas9 protein to the nucleus using the nuclear localization signal)
2. The crRNA and tracrRNA are combined into a single gRNA
ALL you need is 1 protein and 1 gRNA
Diversity of CRSIPR systems
Each types of system has a unique PAM that is recognized
ALSO each type of system employs a different mechanism to destroy viral DNA
- Exception – Types 6 destroys RNA
Each species of bacteria can have multiple CRISPR systems + can have multiple CRISPR arrays within their genome
Why is there so much diversity – CRISPR is an adaptive immune system = requires the capacity for diversity to keep up with the quickly mutating viral threat
Cutting with sgRNA/SpCas9 Complex
SpCas9 uses NGG PAM (NGG PAM must be immediately downstream (3’ end) of the protospacer on the non-target srand)
IF have PAM and the spacer is complementary to the target then the SpCas9 creates a dsDNA break 3 nucleatides upstream (5’) of teh PAM
Answer – B
NOTE – Need GG on the 5’-3’ strand (NGG is on non-target strand) + Need target sequence to be complementary to the spacer
- Cut = 3 nucleotides upstream of the PAM
Cas9 will only target if have the correct PAM and there is no mismatch between the spacer (crRNA?) and the target sequence
Off-traget cutting
SpCas9 is highly selective BUT it can cut off-target sites
- Cas9 can tiolerate small mismatches that are far away from the PAM
Overall - As you travel farther from the PAM = complementarity requirement for base pairing is reduced –> Small mismatch on the 5’ end can be tolerated (I THINK 5’ end of the guide)
Cas9 can have PAM flexibility (NAG(G/C)) –> Cas9 can pair with NAGG and NAGC PAMs with low affinity = leads to eronenous cuts
Beyond Cas9
- Mutated Spcas9 orthologs can improve cutting specificity + can change the PAM sequence + can change cutting behavior
- Example – Cas9v24 = used NGA PAM instead of NGG PAM –> can be used if the desired cut site does not have NGG site nearby
- Type 2 CRIPSR/Cas systems from other species use different PAMs
- To find PAM diversity = look to other type 2 Cas systems in other species
Since first use SpCas9 has seen improvment modifications
Use of CRISPR in lab
When CRISPR is used in the lab it is often with the goal of making mutations
Can make:
1. Mutations
2. Insertions
3. Deletions
What causes mutations in CRISPR
Cas9/gRNA cleave the dsDNA BUT it does NOT directly mutate the genome INSTEAD mutations are cause by endogenous DNA damage Repair mechanisms
Way that the cells responds to the berak informs the modification in the genome
DNA damage repair mechanisms used in CRISPR
- NHEJ – Direct ligation of blunt ends
- Error prone mechanism –> may generate indels –> Indels = causes frameshift mutation —> Frameshift mutations are used to knockout genes - Homology Directed Repair (HDR) - Templated repair from sister chromatid or other donor DNA
- Can precisiley insert exogenous sequences
- Repairs in faithful manner
NHEJ
During NHEJ a number of proteins are recruited to the site of the dsDNA break –> Recruitment of proteins leads to a insertion or removal of nucleotides before the 2 strands are joined together –> means the processes is often mutogenic
Imprecise repair causes Indels
- If the cut occurs in a protein coding gene and an INDEL occurs –> INDELs will cause a shift in reading frame of the gene = can result in a premature stop codon
What happens in the genome is repaired correctley following cleavge (get the same DNA sequence that had before the cit)
If the genome is correctly repaired THEN cas9 will cut again either the target sequence no longer matches the sgRNA or the PAM is mutated
- Cas9 will cut again unto an INDEL is created that disrupts the target sequence or the PAM
What do Frameshift mutations lead to
Frameshift mutations can lead to a premature stop codon
Premature stop codons can Knock out genes and lead to loss of protein expression in 2 ways:
1. Triggers non-sense mediated decay to degrade nascant mRNA
- Non-sense mediated decay = detects early stop codons and degrades problamatic mRNA
2. Coding for a non-functional protein product
- Even if the mRNA is translated it can generate a truncated non-functional protein
Exercise #1 – Knock out the humal PLK4 gene
Question 1 - Where would you target the sgRNA?
Want to target early in the gene
- Might target exon 2 because you could have an AUG downstream that can be used (If started before that AUG then could also get translation past the second AUG in exon 2 )
When targeting DNA to cuase deletion = want to make sure that you start translating (need to start past the start codon) and THEN have a frameshift = need to be in coding frame in exon 1 or exon 2
- Want to target early because want frameshift to start early on
Can target promoter – gives a LOF BUT probably won’t give a full KO
- Frameshift = full KO
What does Exon 1 start with
Exon 1 does NOT start with ATG (have a 5’UTR in exon 1 –> means you have a start codon in exon 1 BUT not at the very beginning
Exercise #1 – Knock out the humal PLK4 gene
Question 2 - What type of alteration are you looking for?
Indel that is NOT a multiple of 3
Need INDEL not in a multiple of 3 because want a frameshift –> frameshift gives a non-sense protein that will terminate at some point
Exercise #1 – Knock out the humal PLK4 gene
Question 3 - How do you confirm knockout of the intended target?
PCR region and send for sequencing of do a western blot for the encoded protein
IF have a heterozygous deletion –> get mixed signals at edited based when sequence
- Heterozygous deletion = deletion on 1 chromosome but not others
- Homozygous could also have different INDELS in different alelles –> Sequence can look messy during sequencing
Exercise #1 – Knock out the humal PLK4 gene
Question 4 - How do you deal with potential off-target events?
Do BLAST of guide against the whole genome to look for places that the guide could bind OR can use a software to look for off target sites –> can make more specific guides/look to see if the guides are specific
- Can also use multiple gRNA
To check:
1. After you do the KO sequence the rest of the genome to make sure there was no off target sites (look at the sites that were predicted to be off targets and make sure there was no actual off target)
2. Complement deletion –> IF you add the gene back yo should be able to revert to the original phenotype
- THEN can know that the phenotype is specific to YOUR KO of the gene and NOT due to and off target effect
Using multiple gRNA in CRISPR
Each gRNA would have a different targets
IF have 4 guides for 1 gene that all give the same phenotype then can be sure the phenotype is not due to an off target
- Adding in these 4 guides seperatley (KO the gene 4 ways –> know that phenotype is due to KO and not off targets because the odds that all 4 caused the same off target is very low) (CHECK with riana)
HDR-Driven genomic insertions - Example 1 (Inserstion)
Example 1 – Insert the green sequence into the genome
Process - After making the cut with Cas9 –> introduce a syntehtic repair template to trick the HDR machinery into copying over out insertion sequence
- Want the cut to be as close to the insertion site as possible
What is needed to use exogenous tempalte/HDR
- In order to use exogenous template/HDR = NEED upstream and downstream homology arms on the exogenous template DNA
- Need the homology arms to be 30-300 BP
- Homoology arms = signlas to cell that the template can be used for repair - In image – inclusion of the green sequence on the template DNA leads to its insertion into the genome
- Need the homology arms to be 30-300 BP
- Must mutate out PAM or create a silent target sequence mutation to avoid recutting
- To avoid having cas9 cut the repair template OR from recutting the new properly edited genome –> a mutation must be made in the target or PAM sequence or insertion
HDR driven deletion
Depending on size of deletion one or two cuts may be made with cas9 around the desired deletion site
Example #2 (deletion) - Exclusion of the purple sequence in the repair template leads to its precise deletion form the genome
- Template DNA = lacks the purple (introduce a homology template does not contain the sequence that you want deleted)
- Unlike Indels created by NHEJ – this method of insertion and deletion are very precise
Must mutate out PAM or create a silent target sequence mutation to avoid re-cutting
HDR driven mutations
Overall – by doing deletion/insertion paradigns you can make precision mutations using CRIPSR
Example #3 - Cut site close to or within region being mutated
- Introduction of the green mutation in the repair template leads to its precise change in the genome (get the mutation from the template in the final repaired genome)
- Still must mutate out PAM or create a silent target seqeunce mutation to avoid recuting
Avoiding Re-cutting when using HDR
Need to edit the repair template avoid cutting by Cas9
To avoid indels –> repair templates must evadae targeting by Cas9
To avoid indels:
1. Introducing a synonymous mutation in the target sequence
2. Introducing a mutation in the PAM
Need to pick a guide whose target or PAM will be interrupted by the insertion/deletino/mutaion so that the guide can no longer cause cleave at the repair site
Image - repair template has a series of muations (mutations in red) in PAM and in the target sequence that prevents cas9 from binding and cleaving again
- All synonyous mutations = not changing the proteins translated
Delivery of CRISPR/Cas9 into cells
Introducing Cas/CRIPSR systems into cells is a hurdle faced by many reserachers
- Different stradegies have diffrent cutting effciciney and persistence
Delivery stradegies:
1. Viral Transduction – Stable integration of Cas9 and sgRNA sequence into genome
2. Microinjection/Electroportaion of RNP/sdRNA/Cas9 - Transient burst of Cas9 activity
3. Transient Transfection of CRISPR/Cas DNA or mRNA – Burst of activity
- NOT incorporated in the genome
Need to consider imporatnt edtting efficincey is + how well cells tolerate the delivery method + if want stable incorportion of the CRIPSR cas system into the genome for persistent cutting
Nuclease Dead Cas9
Nuclease Dead Cas9 - kills the nuclease activity of cas9 with mutations in the Cas9 sequence
- Can tag dCas9 with effector domains
dCas9 can be fused to other function proteins to:
1. Tag genomic loci with a florecnet marker –> used for localization module in the genome
2. Precise random mutation with base editors
3. Edit the epigenome such as methylation status or histone methylaton
4. Activate or repress transcripton (CRISPa/CRISPRi)
How does cas9 scan DNA
Cas9 scans DNA 3’-5’ and looks for PAM at the 3’ end and binds to the 5’-3’ strand
Answer – B (44%) –> because need indel of ½ (2/3) and need 2 events because need both alelles = 2/3 X 2/3 (because indepennt) = 4/9 = 44%
Example - Designing an HDR template for knock in of an AID tag
gRNA overlaps with the stop codon where you introduce the AID tag –> now that you have tag = guide no longer matches the target = cas9 doesn’t cut again = have stable edit
See in image (A) - see gRNA overlaps with stop and AID is introduced at the cut = end gRNA no longer matches = cas9 can’t recut
How do you make the HDR template
To make HDR repair template –> can have plasmid that has AID tag –> Use primer that amplify the AID tag
IF want to add the tag to 20 genes – use 20 primer pairs and each primer will have different homology arms for the gene of interest (IMAGE – light blue is the homology arms)
- Homology arms are part of the primer = adds homology arms upstream and downstream of the AID tag)
- 3’ end of the primer (both foward and reverse) stays the same –> amplifies the AID tag (dark blue in image)
END – get 2 recombination events using the homology arms to insert the tag into the right spot
Tags that you can insert using HDR
- Epitope tag
- Use when doing a western – if don’t have an Antibody for protein NOW can do a western and have an Antibody bind to epitope tag
- GFP – allows you to see where cas9 localizes in cell
- Selectable tag –> allows you to select for cells that received your editing construct
Identifying editted clones
Overall – separate edited cells to individual cells on a plate –> then grow to get clones in each well
- Can separate to individual cells using a robot or by diluting so that you only have 1 cell per well)
To check using PCR (can see in heterozygous or homozygous):
- IF have no edit – get same size bad
- IF have edit get bigger band on gel (because adding something in) ; IF edit both alleles = bigger bands
- Heterozygous (edit 1 allele) = see 2 bands (1 band is the WT and 1 band is edited)
Looking for editted clones
Can use PCR or western blot
Western Blot:
- See WT band
- Heterozygous - have WT band and a larger band
- Homozygous edited - have a larger band (because aded something to protein)
IF there is no antibody for the gene of interest you can add an HA tag
- WT has no signal (because not edited = doesn’t get HA aded = no signal )
- Edited - have a single when use HA AB
- Heterozygous - has half and half (small size change compared to homozygous = hard to tell between homozygous and heterozygous )
Potential issue in HDR
Issue – if you want to insert a AID tag next to stop (red next to stop in image) BUT there is no PAM THEN you need to cut more upstream (cut where the scissors are)
IF the guide matches the blue sequence (once the template is inserted in) THEN need a way to prevent cas9 from continuing to cut once the edit is made –> To do that = introduce mutations in the tag (template?) that prevents the gRNA from perfectly matching the cut site (blue recode in image)
- Recoding the region that is also normally present in the WT
IF making a cut where the scissors are then gRNA matches that blue region where the cut is = when add the Red AID then blue would still match the gRNA = cas9 would recut BUT since recode it = cas9 won’t cut again
- Recode the region = now have a mutant every 3 BP –> once the edit is introduced cas9 stops cutting and you only have the insertion
OVERALL recoding and cut site
IF the cut site is where you want to insert the tag = woudn’t need to recode because the tag would overlap with the cut site and the cut site would be chnaged = gRNA can’t bind
IF the tag doesn’t overlap with the cut site = need to cause change in cut site (recode) so cas9 doesn’t cut again
- When you cut more upstream than the insertion site (cute where scissor is but the red is inserted more downstream) = need t screw with the repair template template so that cas9 doesn’t re-cut
- Tag insertion does not screw with cut site when cut is more upstream than insertion site
How does the template actually get inserted into the target DNA
Overall - Need 2 recombination events to insert the template into the target (1 event in the 5’ end and 1 event in the 3’ end)
Need to force 1 of the recombinatiion events to be downatream enough to insert the tag
- No recoding = have recombination in the 5’ and too far upstream on the 3’ end (to far upstream and won’t insert the tag) –> THERFORE have recoding = causes corect recombination (at the 5’ end and further downstream at the 3’ end where want insertion of tag)
In image - recoding favors recombination in the 2 green areas = get the red tag right next to the stop codon (check with riana)
Answer – B., C, D,
- Don’t want to cut he template (mutations stabilize the template)
- Prevents INDELS (indels would happen if you were able to keep cutting because eventualy would repair with NHEJ)
- Stabilizes the HDR template
Example - Knocking in a mutation adding L89G mutation
Example - adding L89G mutation
In image – red is PAM and underline is where the gRNA binds ; bottom is the WT allele
Target site = recombines with the template DNA that you gave –> PAM is now disruption AND introduced the mutation that you wanted to make (L89G mutaion – see have Leu –> Gly)
- PAM in WT = TGG in red ; in mutant have TGT = disrupted the PAM when added in template
- ALSO made AF3 mutation –> used to screen cloned (adds in a RE cutting site)
End – template adds the mutation + disrupts the PAM = means cas9 won’t keep cutting the sequence because the DNA won’t match the gRNA and because the PAM was mutated
How do you check for successful knockin of a point mutation (look to see if the edit is made)
To look to see if the edit is made:
Take each clone –> do PCR –> could sanger seqeunce OR can cut PCR amplicon using RE
- In PCR the size of the band is the same in WT and edited because have 1 BP change
Because you added the restriction enzyme cutting site –> NOW can verify if have edit:
- IF Restriction enzyme cuts the PCR amplicon into 2 peices = then know that the edit was made (becaue know have RE sites = know edit was made)
- If get a shorter product = edit was made vs. Longer product = WT vs. Both size bands = heterozygous
Answer – D –> want to cut as close to edit site as possible
Cutting close = disrupts the target + increases the frequencey of correctley editted clones (because reocmbination is going to happen close to the cut site = if cut site is far from edit you can have recombination occur but not get edit into DNA)
Be mindful of what you repair template is going to be because it might get recut unless you do something to prevent recutting
- Want to preserve the amino acids sequence of gene (do mutation in 3rd base of each codon)
Activity #5 – Make a large genomic deletion (delete PL4 gene) - How would you delete PLK4
To delete PLK4:
1. Hope that you will get an INDEL with NHEJ
2. Can make a repair template –> fuse homology regions to random DNA that has RE site in it
To do option 2:
1. Have 2 guides (2 guide cuts upstream and 1 guide cuts downstream) = cut out the middle DNA
2. After cut with 2 guides:
- Can stick the ends together with NHEJ
- OR can use repair template and incorporate RE sites and do PCR and see if it is there or not using a restriction digest
Answer – A
Cas9 + base editer
Use gRNA/cas9 to get cas9 to the gene of interest BUT insead of cutting and repairing we can use a base editor
- Mutate both nuclease domains = get dead cas9 = won’t cut site
Can fuse a cytodine demainae with cas9 –> once cas9 binds to DNA it will make the R loop with 1 free strand of exposed ssDNA –> demainase fused to cas9 will cause Cytosine to be deaminated to a Uracil (C –> U) –> U will pair with an A (instead of a C-G) so when DNA is replicated the U is matched with A = NOW made an edit without a dsDNA break
Issue – deaminase is flexible when it is attached to cas9 = can edit all of the C in the area
- Relies on there being a C in the right place + no precise control over what C is edited
Can do the same thing with ADAR enzymes
Using cas9 to make a nick
cas9 can also make a nick instead
IF the cas9 has 1 activated nuclease domain (nickase) = made a ssDNA break on the strand you are not trying to edit –> tells cells to repair the top strand –> Cell chews up the top strand and repairs it using the bottom strand as the template -
- SsDNA break is NOT as mutagenic as a dsDNA break
- ssDNA break happens on the strand that is not edited
- By editting the bottom strand and nick the top strand you bias the cell machinery to replicate over the new mutant base (In image) add that A I
Cytosine and adenosine deamination
Normally a CG can be deaminated from a C to a U –> make a nick and host repair machinery degrades the strand and repairs using U as a template so now you get an A
Even if U is fixed by cells DNA repair system you still get progeny cells that have the mutation
