Lecture #5 - CRISPR Part 1

Question 1

Editting Technology Before CRISPR

Answer 1

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

Question 2

Bacteriophages

Answer 2

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

Question 3

Original Purpose of CRISPR

Answer 3

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

Question 4

How does CRIPSR work in bacterial immunity

Answer 4

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

Question 5

What happens during infection by bacteriophage

Answer 5

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

Question 6

CRISPR (Overall)

Answer 6

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

Question 7

CRISPR/Cas9 System overall (key points)

Answer 7

1. 20 BP target RNA is fused with 76 RNA scafoloed
2. 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
3. Creates a dsDNA break 3 BP upstream from the PAM
4. Breaks repaired by error prone NHEJ or HDR

Image - blue is DNA target ; green is gRNA

Question 8

Where does gRNA come from in CRISPR

Answer 8

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

Question 9

What does Cas9 look for

Answer 9

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)

Question 10

Cas9 Nuclease domains

Answer 10

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

Question 11

Cells repairing the dsDNA break

Answer 11

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

Question 12

Cas9 binding to the DNA

Answer 12

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

Question 13

What do cas genes code for

Answer 13

Upstream of the array = cas proteins themselves
- Cas protein = involoved in new sequence (spacer) integration + CRISPR RNA processing + Interference

Question 14

CRISPR locus

Answer 14

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.)

Question 15

Spacer

Answer 15

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

Question 16

crRNA

Answer 16

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

Question 17

Cas9 Nuclease complex

Answer 17

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

Question 18

tracrRNA

Answer 18

tracrRNA = strcutual element that tethers the crRNA (complex the crRNA and the cas9 protein)

Question 19

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

Answer 19

Cas protein recognzies a PAM sequence downstream of the spacer/portospacer base pairing on the dsDNA

Question 20

PAM

Answer 20

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

Question 21

Classes of CRISPR systsems

Answer 21

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

Question 22

Why use Type 2 CISRP systems in the lab

Answer 22

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

Question 23

SpCas9 in Lab

Answer 23

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

Question 23

Diversity of CRSIPR systems

Answer 23

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

Question 24

Cutting with sgRNA/SpCas9 Complex

Answer 24

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

Question 25

Answer 25

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

Question 26

Off-traget cutting

Answer 26

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

Question 27

Beyond Cas9

Answer 27

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

Question 28

Use of CRISPR in lab

Answer 28

When CRISPR is used in the lab it is often with the goal of making mutations

Can make:
1. Mutations
2. Insertions
3. Deletions

Question 29

What causes mutations in CRISPR

Answer 29

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

Question 30

DNA damage repair mechanisms used in CRISPR

Answer 30

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

Question 31

NHEJ

Answer 31

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

Question 32

What happens in the genome is repaired correctley following cleavge (get the same DNA sequence that had before the cit)

Answer 32

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

Question 33

What do Frameshift mutations lead to

Answer 33

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

Question 34

Exercise #1 – Knock out the humal PLK4 gene

Question 1 - Where would you target the sgRNA?

Answer 34

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

Question 35

What does Exon 1 start with

Answer 35

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

Question 36

Exercise #1 – Knock out the humal PLK4 gene

Question 2 - What type of alteration are you looking for?

Answer 36

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

Question 37

Exercise #1 – Knock out the humal PLK4 gene

Question 3 - How do you confirm knockout of the intended target?

Answer 37

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

Question 38

Exercise #1 – Knock out the humal PLK4 gene

Question 4 - How do you deal with potential off-target events?

Answer 38

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

Question 39

Using multiple gRNA in CRISPR

Answer 39

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)

Question 40

Mismatches far vs. close to the PAM

Answer 40

Mutations (mismatch between DNA and gRNA) that are far from the PAM can disrupt binding BUT if a mismatch (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)

Question 41

HDR

Answer 41

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

Question 42

HDR in CRIPSR

Answer 42

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

Question 43

HDR-Driven genomic insertions - Example 1 (Inserstion)

Answer 43

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

Question 44

What is needed to use exogenous tempalte/HDR

Answer 44

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

Question 45

HDR driven deletion

Answer 45

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

Question 46

HDR driven mutations

Answer 46

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

Question 47

Start at Format of donor template DNA