Chapter 15

Question 1

Reverse Genetics

Answer 1

• Begins w a gene sequence, mutate, and seeing impact on phenotype
• Common reverse genetics tech to change a gene in model organisms
• Being specific + intentional with the gene that is modified

Question 2

Common reverse genetics approaches in model genetic organisms

Answer 2

E.coli: knockout mouse by homologous recombo

But more complexity in organism means more complex tech

Question 3

Yeast

Answer 3

• Diff from bacteria, linear chromosomes, competent at picking up circular plasmids
• Weakens the cell wall and allows them to pick up the plasmids

Question 4

Homologous Recombination

Answer 4

• Use endogenous mechanisms of recombo to integrate exogenous DNA fragments into the genome (efficient for organisms such as bacteria, archae and some simple eukaryotes but not very effective for more complex eukaryotes like plants and animals
• Organisms alr have capability/proteins that allows them to echange DNA material, as long as segment thats homologous/shared will align witht he chromosome in the hos species

Question 5

Knockout Library

Answer 5

Collections of mutants in which most/all genes have been mutated by inctivation/knocking out expression, causing loss of function

Question 6

Circular vs linear DNA

Answer 6

• Researchers can weaken yeast cells walls to uptake a plasmid, but will engineer it w a target gene thats homologous to a gene that alr on yeast chromosome only with desired knockout and a selectible marker that will help identify it and create enough of a diff to ensure that recombo is not happening not smash in the middle of the gene and rather flank
• Target gene shares similarity with the target gene on the yeast chromosome
• Bc region on plasmid is homologous to region on the linear chromosome, they’re gonna line up and try to participate in recombo

Question 7

Single Crossover Event

Answer 7

All contents of plasmid get integrated into linear yeast chromosome, and the linear it has the knockout and target gene

• The entire molecule of circular DNA is integrated into the yeast w/o loss of genomic DNA which will disrupt the target gene, making it non-functional.

Question 8

Double Cross over event

Answer 8

Complete integration of the target gene into the yeast chromosome

• Replacement of the target gene with the gene thats in the plasmid

Question 9

How does reverse genetics homologous recombo differ from meiosis?

Answer 9

No exchange of genetic material from the bacterial DNA to the plasmid, more about integration or replacement within a genome for gene manipulation purposes.

Question 10

Knockout Gene Linear Ver

Answer 10

Introduce a short piece of linear DNA that shares a homologous region with the yeast DNA

Question 11

Knockout Gene Linear Ver Single Crossover event

Answer 11

Integration of desired gene into yeast chromosome but lose end of the chromosome, becomes an acentric fragment and you might get successful loss of functon, but it could be due to the acentric fragment or bc of sucessful integration

Question 12

Knockout Gene Linear Ver Double Crossover event

Answer 12

More desirable because don’t lose genetic information.

Question 13

What is the method of choice for most homologous recombo experiments?

Answer 13

Linearized DNA molecule recombine at a higher Freq than circular ones so it’s the method of choice

• Bc sometimes single crossover recombinants are lost bc the loss of genetic material on the acentric fragment results in lethality, helps ensure the more precise double crossover insertion occurs more iften

Question 14

Reverse Genetics Approach #2: Random insertional mutagenesis followed by gene-specific screening

Answer 14

• Some organisms have very well documental transposable elements
• Create an insertion library and encourage random insertions of documented transposable elements and use gene and element end primers to detect whether it landed inside of a gene or didn’t

Question 15

How do u create a library?

Answer 15

• Use a transposon and encourage them through expression of transposase to be inserted on almost every single gene of interest

Question 16

Whats the diff from the targeted approach of recombination and random mutagenesis

Answer 16

Allowed to be jump how they want

Question 17

Gene-specific Screening

Answer 17

• Use PCR, and design primers that go in the fwd + rev direction for gene primers and gene primers and transposable elements primer

Question 18

What do u see if transposable elemnts didn’t land inside a gene?

Answer 18

• If transposable elements did not land inside a gene, then going to have a band only where the fwd and rev gene primers were able to align and amplify the DNA and it could travel thru the gel

Question 19

What do u see if the mutant is inserted inside of a gene?

Answer 19

• If mutant is inserted inside of a gene, then see that gene primers were able to align but also transposable elements primers also align so band for each direction.

Question 20

Reverse Genetics Approach #3: Gene silencing by double-stranded RNA

Answer 20

• A dsRNA is a signal for cell to initiate immune response

Question 21

DsRNA Delivery route (3 routes)

Answer 21

1a) Transfect/inject dsRNA

1b) Viral delivery of dsRNA

1c) express a hairpin from a transgene with an inverted repeat -> Makes intracellular dsRNA

Question 22

Dicer Processing

Answer 22

Downstream process once signaled: RNase III enzyme Dicer cuts long dsRNA into 21-24 nt small interefering RNAs (siRNA)

Question 23

RISC Loading

Answer 23

One siRNA strand (the guide) loads into RISC (RNA-induced silencing complex), passenger strand discarded

Question 24

Argonaute

Answer 24

Core protein of RISC

Question 25

DO we need both strands of the siRNA?

Answer 25

No only need one and the other one is discarded

Question 26

Targeting and Silencing

Answer 26

Guide siRNA base-pairs with complementary mRNA; argonaute "slicer" cleaves mRNA (guide siRNA will help RISC find and intercept the intended mRNA

Question 27

Why is the gene silencing by dsRNA an immune response

Answer 27

Endogenous sys alr existing in cells and r trying to protect itself from viruses where the mode of infection is DNA based, so in order to prevent tarnsicription of the mRNA, dsRNA acts as a memory that binds w the harmful phage introduced DNA, cleave it and prevent iit from infecting, will be able to remember the sequence, regulatory mechanism for DNA which u may not want to be expressed all the time

Question 28

Reverse Genetics Approach #4: Crispr Cas9

Answer 28

- Researchers identifiied repetitive elements in some bacterial organisms, bw repeats were novel bp that also change as environment changes - Defense against invading nucleic acids - Adj related genes: CRISPR-associated cas genes including a DNA endonuclease and RNase

Question 29

How does CRISPR-Cas9 Work

Answer 29

- Repeat sequences and unique spacer sequences - Acc crispr elements get transcribed into a non-coding precursor crRNA - Post transcrip processing of precursor involves cas-encoded Rnase (processes the precursor crRNA into individual repeat unit crRNAs)

Question 30

Tracer RNA

Answer 30

- Tracer RNA gene also transcribed into a small noncoding RNA - One part binds to cas endonuclease and another part binds to crRNA, creating RNA-protein complex

Question 31

tracer-RNA- crRNA-Cas complex

Answer 31

Capable of introducing double stranded breaks in invading DNA molecules (phage/plasmid) at sites determined by the crRNAs (regions homologous to the crRNAs)

Question 32

Spacer sequences

Answer 32

- Cell has memory, if attacked by phage that had dsDNA and survived, that unique repeat and spave sequences directs CRis to the dsRNA so it can be destroyed

Question 33

Crisr re-engineered for genome editing

Answer 33

- 2 functional components of this system are: 1) Guide RNA that binds to a target dsDNA sequence 2) cas9 an endonuclease that cuts the target DNA sequence

Question 35

What does the guide RNA contain?

Answer 35

CRISPR RNA and TracrRNA - These components join tgt at the location of the gudie RNA binding and cas9 cleaves DNA, creates a complex the RNA that we want to introduce has reocgnition of where the dsDNA the homologous strand is to that,

Question 36

Two mechanisms available to repair cleaved DNA

Answer 36

- when ds break happens cell will want to repair itself through NHEJ and Homologous Recombination - If you want to target a specific area, will decide what that RNA/ guide sequence is gonna be to locate the corresponding region in the DNA, once it gets there and makes the cut, depending on where it is in the cell cycle, It will in engage in NHEJ or homologous recombo - Homologous recombo requires the cell to be in a certain stage in the cell cycle because it needs the homologus sister chromatids to participate, and if it is not at that point in the cell cycle, it can engage in NHEJ

Question 37

What do we typically have when u have NHEJ?

Answer 37

- Deletion events, not perfectly blunt ends and will lose nucleotides when making them blunt

Question 38

What do you do if the goal is to only remove a certain base pair?

Answer 38

- If all you wanna know is excise a harmful codon - Don't have to send in replacement with the guide, just have RNA cleave and NHEJ ligate the ends tgt, successfully create a knockout

Question 39

What do you do if you want to incorporate donor DNA?

Answer 39

- Send in DNA that u want and incorporate it thru homologous recombo.

Question 40

CRISPER-Cas9 Applications and Ethical Considerations

Answer 40

Getting Crispr cas9 into a cell: - Transgenes, injection of cas9 protein and guide RNA - Multiple guide RNAs to target multiple loci - CRISPR-Cas9 broadly applied to study gene therapy agriculture