Lecture #5 - CRISPR Part 1 Flashcards

Question

Cutting with sgRNA/SpCas9 Complex

Answer 1

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 2

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

Answer 3

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

Answer 4

1. 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 2. 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

Answer 5

When CRISPR is used in the lab it is often with the goal of making mutations Can make: 1. Mutations 2. Insertions 3. Deletions

Answer 6

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

Answer 7

1. NHEJ – Direct ligation of blunt ends - Error prone mechanism --> may generate indels --> Indels = causes frameshift mutation ---> Frameshift mutations are used to knockout genes 2. Homology Directed Repair (HDR) - Templated repair from sister chromatid or other donor DNA - Can precisiley insert exogenous sequences - Repairs in faithful manner

Answer 8

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

Answer 9

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

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

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)

Answer 17

Mutations (mismatch between DNA and gRNA) that are far from the PAM can disrupt binding BUT if a mismatch is close to PAM (between gRNA and DNA) won’t get any binding - MEANS mismatches are more tolerated at the 5’ end of the guide sequence Cas9 = looks at the PAM and then looks at the 3’ end --> Because of this cas9 can still cause a dsDNA break even if the guide doesn’t match the DNA towards the 5 end (farther from the PAM)

Answer 18

HDR = templated repair mechanism that precisely repairs the genome HDR uses DNA that is homologous to the cut DNA (The homologous DNA acts as a template for repair) - Often uses the sister chromatid as the template - Repair by HDR results in a clean exchange on DNA from the template = get a perfect repair

Answer 19

IN CRIPSR - introducing an exogenous repair template Can result in: 1. Precision insertions or 2. Deletions or 3. Modifications can be achieved In CRISPR = need to provide the repair template that includes the edit that you are trying to make - Template repair can be exploited to make precise edits

Answer 20

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

Answer 21

1. 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 2. 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

Answer 22

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

Answer 23

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

Answer 24

Depending on the need – template DNA may come in many formats What affects repair efficiencey: 1. Template stability in the cellular environment 2. Access by HDR machinery Resercahers balance stability and cytotoxicty with how easily HDR machinery can access the DNA for template repair

Answer 25

1. ss/dsDNA oligio (small mutations) 2. Plasmid DNA (large insertions/deletions) 3. Linearilized plasmid DNA (large insertions/deletions) - Linearization improves access to DNA as a template BUT reduces stability

Answer 26

Repair templates may include special sequences to aid in selection/validation Example – resistance genes + Unique RE sites + florurophore + Affinity tag Have many possibilities for genomic deletions and insertions - Example - can cut out key functional domains + insert degrons

Answer 27

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

Answer 28

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

Answer 29

Many factors affect the Cell’s decision to use HDR or NHEJ when repairing the break cause by Cas9 Factors: 1. Cell Type 2. Cell cycle stage 3. Locus 4. Concentration and format of donor template (ex. SsDNA vs. DsDNA vs. Plasmid vs. Linear) 5. Length of homology arms 6. Position of the cut relative to the edit HDR is generally much less efficient than NHEJ

Answer 30

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)

Answer 31

How you get Cas9 and gRNA into cell type of interest is where a lot of limitation exists

Answer 32

Cas9 scans DNA 3’-5’ and looks for PAM at the 3’ end and binds to the 5’-3’ strand

Answer 33

1. Amount of labor/money/time it would take 2. Efficiency of delivery 3. Speed at which the Cas9 starts cutting 4. Window of time the cutting occurs (how fast does it occur and how long does it occur)

Answer 34

1. Assemble the Cas9/sgRNA Ribonuloprotein complexes 2. Use a sgRNA and an mRNA encoding Cas9 3. Clone the sgRNA + Cas9 into a plasmid 4. Clone the sgRNA + cas9 into a plasmid and generate a lentivirus

Answer 35

Overall - Pre-incubate cas9 with the gRNA and make sure loading of the gRNA happens - Get RNP into cells using electroporation or lipofections (transfection or transduction) - Transient Pro - makes sure loading of the gRNA happens + ready to cut instantly because preloaded with gRNA + Faster because Cas9 is alreayd translated and bound to the guide Con - complex is very big = it will be hard to get into cells

Answer 36

Transient because the mRNA will eventually be degraded - How ever much protein and RNA you give is what you get until they are dgeraded Slower than RNP because need to translate cas9 BUT could give you a longer editing window than RNP (if stability of the mRNA is longer than RNP)

Answer 37

Less transient because DNA stays longer than RNA and protein - Plasmids are not replicated in mamalian cells = plasmid will stay for days but NOT forever - Plasmid will get diluted over time because it is not replicated Pro - Allows you to select for cells that receive cas9 because can have a selectable marker on the plasmid (can kill cells that did not receive plasmid)

Answer 38

Lentivrius is an RNA virus that is RT to DNA and the DNA is integrated in to the genome of the host and then the DNA is transcribed and translated - Delay in making cas9 BUT the host makes cas9 and the guide for as long as it lives - Stable integration = get stable cas9 cell line or animals that express cas9 forever - Has the longest window of editing Pro - Lentivirus = gets good packaging effcicincey = deliver more of cas9 to target - Cells are well targeted by the virus

Answer 39

Longer window of editing = more chance of off targets Compared to transient where there is less time for cas9 to find off targets

Answer 40

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%

Answer 41

In C terminus at the end of the protein (right in front of the stop codon) - Might have sequences at the N terminus that you don’t want to mess up = put the AID tag at the C terminus Add before the stop codon because you want to make sure it gets translated + Add at the end because then it is less likley to influence the translation of the rest of the genes Theoretically - Could add internally in the protein IF you know that there is a flexible linker then you may be able to add a tag in the linger (requires knowing a lot about protein structure)

Answer 42

Right before the stop codon AND you want to make sure it is in frame

Answer 43

Want to cas9 to cut close to where you are inserting your tempalte (close to the edit) WHY cut close - The break will disrupt the target site so once the edit is made cas9 won’t cut again - IF the edit is far from the cut site then cas9 can cut again (because when repair cut get the original DNA that gRNA matches) - IF the cut is in the middle of the insertion site NOW cas9 can’t recut IF Cas9 cuts DNA and repairs the DNA and repairs the DNA back to WT then cas9 will keep cutting until an edit (likely an INDEL) is made

Answer 44

IF inserting tag at the C terminus BUT thee is no NGG near the site THEN you need to cut somewhere different from where you are making the edit --> means that the edit will not disrupt the cut site (because edit is now far from the cut site) = NOW cas9 can cut again

Answer 45

NEED homology region on either end of the template/tag that you are inserting Homology arms go at the 5’ and 3’ ends --> allows the cell machinery to add the foreign DNA to the endogenous locus - In HDR = Relying on the cell to recombine at the upstream and downstream regions (at the homology arms) = need 2 recombination events to insert the DNA

Answer 46

Goal - Add Exogenous DNA with the tag and homology arms upstream and downstream of the tag Synthesize the template and give to the cells Template given can be on: 1. Amplicon --> PCR template and electroporate to cells (linear DNA) 2. Add plasmid with template

Answer 47

HDR template needs homology arms upstream and downstream of the tag

Answer 48

1. IF using a plasmid --> add a selectable marker and spatially separate the cells on the plasmid 2. Use Machine that separates cells into wells and grow in liquid so each well in the plate came from 1 cells

Answer 49

Use western blot for protein product or Sequence

Answer 50

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

Answer 51

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

Answer 52

1. 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 2. GFP – allows you to see where cas9 localizes in cell 3. Selectable tag --> allows you to select for cells that received your editing construct

Answer 53

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)

Answer 54

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 )

Answer 55

1. Cut close to the insertion point (less than 30 BP) 2. Homology arms = 35 BPs 3. Recode (wobble) the sequence between the insertion point and the cut 4. Include mutations that prevent cutting of the edited locus by cas9 5. Use a high-molarity of PCR amplified DNA

Answer 56

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

Answer 57

Recode at the wobble position of each codon) = not changing the AA --> make the same protein but because DNA is recoded gRNA can’t bind - END - Have a mutant every 3 BP

Answer 58

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

Answer 59

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 60

Answer – B., C, D, 1. Don't want to cut he template (mutations stabilize the template) 2. Prevents INDELS (indels would happen if you were able to keep cutting because eventualy would repair with NHEJ) 3. Stabilizes the HDR template

Answer 61

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

Answer 62

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 63

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)

Answer 64

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 65

Could get HDR with the Sister chromatid could happen BUT cas9 would keep cutting until you get a mutation

Answer 66

Answer – A

Answer 67

Normally cas9 = gives a dsDNA break --> dsDNA break allows for HDR process to initiate Issue – dsDNA break can be dangerous because it can be mutenogeic - Can get mutation due to off-target cuts + dsDNA break stresses the cells - Some cells might not like the DNA damage --> cells could die - Off target effects could be bad because you could eliminate a tumor supressor --> even at a low rate in a patient this could cause cancer Solution – can edit with cas9 technology that skips dsDNA break

Answer 68

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

Answer 69

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

Answer 70

Issue - If U is added to DNA the cell will try and fix it IF you don’t want to fix the uracil added to DNA (want U to stay so DNA oppoiste it gets miscoded) --> add a UGI which inhibits the DNA repair machinery that would normally try and repair U to C NOW - Fuse cas9 to the 2 proteins domains (cytosine deaminiase and the UGI) --> 1 makes the edits and 1 inhibits the DNA repair machinery

Answer 71

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

Lecture #5 - CRISPR Part 1 Flashcards

(100 cards)