Lecture 4 - RNAi and Gene Editing

Question 1

What is classical/forward genetics?

Answer 1

Identify random mutations and look for mutants that caused the change in phenotype so the genotype can be studied

Question 2

What is reverse genetics and how can it be carried out?

Answer 2

Using targeted approaches on a gene sequence to make mutations so the phenotype can be studied
- Partial gene knockdown by RNA interference (reduces gene function)
- Gene knockouts via CRISPR/Cas9 (alters gene function by gene editing)
- Altering a gene in vitro by site-directed mutagenesis

Question 3

What does an anti-sense RNA do?

Answer 3

Anti-sense RNA is used to knockdown genes by injecting RNA that is complementary to a specific mRNA that binds to prevent the translation of the mRNA

Question 4

What is a common control in anti-sense RNA experiments?

Answer 4

Adding a sense-control/oligo which has the same sequence as mRNA so it will not bind and interfere with translation to give it a knockdown phenotype - serves as a negative control

Question 5

How can the amount of knockdown by anti-sense RNA be reduced?

Answer 5

By adding increased amounts of sense oligo - reduced the effect of anti-sense oligo because the sense-oligo competes with mRNA to bind with it *Was thought to happen

Question 6

What do combining sense- and antisense-oligos result in?

Answer 6

Over 100x stronger knockdown than antisense alone (discovered by Fire and Mello in the late 1990s)

Question 7

What is RNAi

Answer 7

A method to knockdown and reduce gene function that has several related pathways that use related sets of enzymes and differ in outcome (translational repression or RNA degradation)

Question 8

Why is RNAi referred to as a knockdown?

Answer 8

Because it is not a genetic technique - we’re not changing the DNA, we’re altering the expression of the gene (e.g., at the level of RNA degradation, it does not usually remove all of it, which often only causes a partial effect)

Question 9

In terms of processing, how do small noncoding RNA transcripts regulate animal and plant genes by RNAi?

Answer 9

Dicer complex cleaves dsDNA from a virus into 21-23 nucleotide fragments called small interfering RNA (siRNA) and the RISC/Argonaute complex cleaves one strand of siRNA and leaves behind the guide strand (RISC complex + ssRNA)

Question 10

How did RNAi likely evolve to exist in all animals?

Answer 10

Likely evolved as a form of immunity against viruses and transposable elements in our genome

Question 11

What is shRNA?

Answer 11

Small hairpin RNA (shRNA) is due to RNA produced by many viruses and transposons folding back and hybridizing to itself

Question 12

How is shRNA processed?

Answer 12

Dicer processes shRNA to generate siRNA, which can then be handed off to the RISC complex where the passenger strand is cleaved and removed so you’re left with the guide strand associated with RISC

Question 13

In terms of CRISPR/Cas mediated immunity, what was discovered in 1987?

Answer 13

1) CRISPR locus was found in many bacteria which contained regular repeating palindromic sequences that were conserved in all bacteria that had them with an unknown purpose - called repeat sequences
2) Seemingly random sequences found between the repeats and were not conserved - called spacer sequences

Question 14

In terms of CRISPR/Cas mediated immunity, what was discovered in 2005?

Answer 14

Sequencing of spacer sequences revealed homology to sequences from various bacteriophages and plasmids

Question 15

In terms of CRISPR/Cas mediated immunity, what was discovered in 2006?

Answer 15

Cas (CRISPR associated) genes were typically found near the CRISPR array and encoded enzymes with predicted activity

Question 16

What is the most important Cas gene and why?

Answer 16

Cas9 because it binds to CRISPR RNA (cRNA)

Question 17

In terms of CRISPR/Cas mediated immunity, what was discovered in 2007?

Answer 17

CRISPR array is transcribed
- Array of spacer elements is transcribed to produce a large RNA called pre-CRISPR RNA (pre-crRNA), in which a number of proteins encoded by Cas genes processed to produce smaller crRNAs

Question 18

In terms of CRISPR/Cas mediated immunity, what was discovered in 2008?

Answer 18

1) Presence of a given phage spacer sequence in its CRISPR array correlates with resistance to infection by that phage
2) Mutations in Cas genes abolish “immunity” to bacteriophage infections if they have similar sequences to one of their spacer elements

Question 19

What are the important elements for type II CRISPR?

Answer 19

1) CRISPR Array - Sequence of spacer elements derived from viral genomes that invaded the cell at some point
2) Spacer and repeat elements
3) Cas genes - Cas1 and Cas 2 genes function with Cas9 genes in the first steps of the immunity system by processing incoming viral DNA, and Cas9 also has a later role where it associates with crRNA to mediate target recognition and cleavage
4) Tracr Gene - Noncoding RNA with region that is complementary to CRISPR repeat sequence (transcribed gene and functional as RNA but doesn’t make proteins)

Question 20

What makes up crRNA?

Answer 20

CRISPR repeat and spacer

Question 21

What makes up the guide RNA?

Answer 21

crRNA and tracrRNA

Question 22

How is type II CRISPR carried out?

Answer 22

1) Virus that has never infected the cell before invades the cell and injects its dsDNA into the cell
2) Cas9 protein recognizes the invading DNA and a very short sequence of 2-3 nucleotides (PAM sequence), which is randomly found on viral or plasmid DNA
3) Cas 1 and Cas2 endonuclease and Cas9 function together to cleave the viral DNA immediately upstream of the PAM sequence, and then again approximately 35 nucleotides further upstream
4) Results in a 35 nucleotide long sequence called the spacer element (lacks PAM sequence) which is inserted into the CRISPR locus of the bacterial genome between two CRISPR repeat sequences
5) CRISPR locus is transcribed to produce a single pre-crRNA which includes spacer elements from the invading virus and those already there
6) Pre-crRNA is processed into a smaller piece of crRNA that contains a single spacer element and its adjacent CRISPR repeat sequence which gets put into the Cas9 protein
7) tracrRNA which has a complementary sequence to the CRISPR repeat from tracr gene is also incorporated into Cas9
8) crRNA is ready to face newly incoming viruses and viral DNA

Question 23

What happens the next time a virus infects when there is a mature CRISPR/Cas9 armed and ready?

Answer 23

• crRNA/Cas9 complexes recognize the incoming DNA because the crRNA in the cas9 has complementarity to one of the dsDNA - allows Cas9 to rapidly cleave the incoming viral DNA
• Recognition only occurs if the spacer element in crRNA has a complementary sequence to the viral dsDNA and if a 3’ PAM sequence on the invading genome is just downstream of it

Question 24

What kind of immunity does CRISPR/Cas9 provide?

Answer 24

Adaptive immunity analogous to the kind of immunity we have against foreign invading bacteria using antibodies

Question 25

How does the CRISPR/Cas9 system avoid recognition of self or autoimmunity?

Answer 25

1) Cas 1, Cas 2, and Cas9 have a strong preference for DNA ends - The bacterial genome will have millions of copies of the PAM sequence that are not recognized and processed into spacer elements because it is a circular genome
* Viral DNA has exposed ends because of its linear structure
2) crRNA/Cas9 cannot recognize DNA in the CRISPR locus because it lacks PAM sequences since the spacer element incorporated between the two CRISPR repeat sequences the first time did not have one

Question 26

How can CRISPR/Cas9 be used to knockout genes?

Answer 26

1) Design a synthetic guide RNA (crRNA + tracrRNA into ssRNA) with base complementarity to a sequence within the interested gene and identify a PAM sequence of interest
2) Introduce Cas9 gene into cells (transfect/transform)
3) Introduce guide RNA into cells
4) Cas9 will associate with the guide RNA and mediate the cleavage of the genomic DNA site because of the PAM sequence near where the guide RNA is targeting
5) Nonhomologous end-joining (NHEJ) will recognize the double-stranded break and rejoin them
Because NHEJ is an error-prone pathway that causes nucleotide deletions, the gene’s reading frame will be disrupted and you’ll almost certainly get a protein that ends pre-maturely

Question 27

What does in vitro mutagenesis do and how is it carried out?

Answer 27

It deletes or replaces an entire domain or specific amino acids in a protein
- Start with known protein sequence and predicted domains
- Introduce mutant protein into cells or animals to test for function (can test in the presence of an endogenous gene or in cells/animals that lack the function of an endogenous gene)

Question 28

What can be done after in vitro mutagenesis?

Answer 28

Can use inverse PCR afterward and get rid of the original unmutated plasmid based on the idea that the DPN1 restriction enzyme depends on the prior methylation of its recognition site
- Original methylated plasmid from bacteria can be recognized by DPN1and the PCR product does not get methylated and is thus immune to DPN1
Therefore, the original plasmid gets destroyed and the only copy is the transformed one which can be made into multiple copies

Question 29

What is an example of in vitro mutagenesis involving beta-TRCP1, Fbox, and SCF?

Answer 29

1) Amino acid sequence of protein beta-TRCP1 can be put into a database to reveal its domains
2) Fbox domain can be identified and shows to interact with SCF, a ubiquitin ligase protein, in other proteins with the same domain
3) To see if our protein mediates the interaction with SCF we can perform a GST pulldown with beta-TRCP1 and if it interacts with SCF, we can infer that it interacts with the Fbox
4) We can make a mutated version for the Fbox of beta-TRCP1, and if it does not interact with SCF, we have identified that it is important for the interaction

Question 30

What are two ways of using inverse PCR with in vitro mutagenesis?

Answer 30

1) Deletion
- Mutagenic primers with no complementarity at a certain part are used and where they start determined the genes that get deleted
- PCR product is linear DNA that lacks the deleted section which can then be ligated to form a plasmid
2) Substitution
- One of the mutagenic primers contains part of a sequence that is not found on its template to be incorporated into the DNA, but there is enough base pairing so that the 5’ end can recognize the 3’ end of the template
- PCR product is linear DNA with a substituted section that can be ligated to form a plasmid
* In both cases, the plasmid can be transcribed and grown-up

Question 31

What orders can proteins be studied in?

Answer 31

1) Clone
2) Epitope/Affinity tag
3a) Express in cultured cells or in vivo
- Immunoprecipitate - identify interacting proteins - mass spectrometry and clone or immunofluorescence, Western blot
- Immunofluorescence, Western blot
- Protein purification - crystal structure or generate antibody
4a) Express and purify from E. coli or in vitro
c) Protein purification - crystal structure or generate antibody
Alt: Fractionate/Assay - protein purification - crystal structure or generate antibody