Forward and reverse genetics

Question 1

What is forward vs reverse genetics?

Answer 1

In forward genetics you start with finding an interesting phenotype and from there search for/identify the gene(s) behind it. “from function to gene”. This is the classic method for studying genetics.

Reverse genetics; start with known gene, assess function/phenotype by altering gene or gene expression. “from gene to function”

Question 2

What is “genetics”?

Answer 2

Genetics = the study of heredity and the variation of inherited characteristics.

Question 3

Define what a “Gene” is.

Answer 3

Gene = distinct sequence of nucleotides that determines the sequence of a protein or RNA molecule
in a cell or virus.

Question 4

What is “phenotype” vs “Genotype”?

Answer 4

Phenotype: an organism’s observable traits, from
Greek “phainein” = “to show”

Genotype: inherited information (in genome).

Basically, you can have a genotype for two separate phenotypes, but the one actually showing is the phenotype.

Question 5

What are forward genetic screens? Name three types.

Answer 5

Forward genetic screens are done to find the phenotype to investigate.

• Spontaneous/naturally occurring variations
• Genetic diseases in humans
• Forward genetic screens: random mutagenesis (to increase probability of occurrence of a rare phenotype)

Question 6

Phenotypes aren’t always visible to the naked eye, give three examples of “molecular phenotypes.

Answer 6

• lacking a germline
• lacking a specific tissue
• changes in RNA/protein metabolism
• different response to environmental change (conditional mutants).

Your phenotype may be visible if you look in the
right place! Finding out where the protein/RNA is normally found can help to finding the phenotype.

Question 7

The functional effects of mutation is either loss-of-function (LOF) or gain-of-function (GOF). What three types of LOF mutations are there? Explain them.

Answer 7

Loss-of-function mutations can be:

• null/amorphic: total loss of function, eg a deletion (as severe as it can be).
• leaky/hypomorphic: Some expression but not like WT, eg. a mutation in a promoter sequence.
• dominant negative: The mutated form affect the expression of the WT allele too, looks like a mix between leaky and null. Eg. A protein that dimerizes when functioning properly, but in this case the mutated copy can bind to the WT protein too, resulting in it’s loss of function.

Question 8

Gain-of-function mutations can be of two types, which and what do they result in?

Answer 8

Gain-of-function mutations can either be:

• hypermorphic: More of the WT gene product, which doesn’t change function but amount. Can have negative consequences like too much phosphorylation which can lead to cancer.
• neomorphic: New function of the gene product, which can be totally different from WT. Can have both positive or negative consequences.

Question 9

There are three variants of dominance that is not complete dominance, which? Give an example of each.

Answer 9

• Incomplete dominance: eg. that a white+red allele results in pink flowers.
• Codominance: eg. blood types where A and B which are dominant over 0, so if you have A0 or B0 you are A or B. But if you’re heterozygous for A and B your AB.
• Allelic series: When several allele types are dominant over the next step, eg CC dom over cchcch which in turn is dominant over chch etc.

Question 10

Alleles can have a variable phenotype. What is incomplete penetrance?

Answer 10

Incomplete penetrance of alleles is when a phenotype is dominant but the dominant phenotype skips generations, very unpredictable and complex.

Question 11

Another type of a variable phenotype is variable expressivity, what is it?

Answer 11

When an allele has variable expressivity it means that carriers of the allele have varying degrees/traits of phenotype. Also unpredictable. Eg. waardenburg syndrome (autosomal dominant) with four different traits that are unpredictably spread out in different individuals over generations.

Question 12

Explain the term “pleiotropy”.

Answer 12

Pleiotropy means that a wide array of phenotypes result from one gene, eg sickle cell anemia (although most of the symptoms are due to the sickle shape of the red blood cells though.

Question 13

What is a “conditional mutant”?

Answer 13

Conditional mutants have a mutation that cause a different phenotype only in certain environmental conditions, eg temperature sensitive alleles, like a mutation that causes reduced metabolism at high temp for example.

Question 14

Explain the difference between chromosomes and chromatids.

Answer 14

Before replication you have one paternal and one maternal chromosome in a pair, after replication you have two pairs of chromosomes and the identical copy pair of the paternal/maternal chromosome are called sister chromatids. As soon as the chromatids separate in cell division, they’re chromosomes again.

Question 15

During meiosis, when does homologous recombination happen and how?

Answer 15

Homologous recombination happens after replication, when there is two homologous pairs of chromosomes (four chromatids). Chromatids from each of the pair can undergo homologous recombination/crossover between which results in gametes containing a recombinant type chromosome (mix of genetic material from paternal/maternal chromosome).

Question 16

Are all genes independently inherited?

Answer 16

No, genes on different chromosomes are independently inherited, but genes on the same chromosome are linked and the closer together they are, the higher the possibility of them being inherited together. So, the distance between genes is proportional to the frequency of the being inherited together.

1% recombination = 1 map unit or 1 centiMorgan (cM). Also, recombination is not even over the chromosome, as there are recombination hotspots with higher recombination frequency, so even though genes are far apart, they can have a high recombination frequency.

Question 17

In a forward genetic screen, the origin of the mutation can differ. Which origins of mutation are there?

Answer 17

Mutation can be spontaneous or induced through mutagenesis. Mutagenesis can be performed by chemicals (like EMS), irradiation (like X-ray or UV) or insertion (like transposons or T-DNA).

Question 18

How does mutagenesis with EMS work?

Answer 18

EMS is a chemical mutagen that donates an ethyl group to Guanine –> O-6-Ethylguanine which behaves and is read like an Adenine resulting in a G-C to A-T mutation.

Question 19

Give an example of how mutagenesis with irradiation works.

Answer 19

Applying UV light to organism –> UV lights hit DNA and case dimerization of bases/SSBs/DSBs –> recognized by DNA repair machinery and when “fixed” results in deletion, insertion or base substitutions.

Question 20

The choice of type of genetic screen depends on the organism and type of mutation. What two things do you need to consider when choosing screen type?

Answer 20

1. Whether the allele is dominant or recessive: F1 screens only work for dominant alleles.
2. Whether the organism can self fertilize or not: F2 screens only work for self-fertilizing organisms.

F3 is the only option for mutations in recessive alleles in non-self fertilizing organisms.

Note: haploid organisms (only one set of chromosomes) have their phenotype visible already so no screen needed.

Question 21

How does a F1 screen work?

Answer 21

In an F1 screen, the allele is dominant and therefore we get the phenotype in the first generation of offspring.

1. Mutagenize sperm cells
2. Mate with WT female
3. Identify dominant mutations in offspring (dominant in 1:1 ratio to WT phenotype).

Question 22

How does a F2 screen work?

Answer 22

Can be used for recessive alleles in self-fertilizing organisms.

1. Mutagenize sperm/oocyte cells
2. Allow F1 individuals to self fertilize (since the organism can self fertilize, the mutation is present in both male and female cells).
3. Identify recessive mutations in F2 offspring (homozygote for recessive trait in 25% of offspring)

If only 1 out of 100 in F2 gen has mutation –> random and not recessive.

Question 23

How does a F3 screen work?

Answer 23

1. Mutagenize sperm cells
2. Mate with WT female
3. Isolate F1 progeny and mate with WT female again to produce separate F2 families (bc it’s not probable to get the same mutation in F1 progeny)
4. Interbreed F2 individuals (both carrying one recessive allele).
5. Identify recessive phenotype in 25% of offspring.

This is quite complicated as you need separation and sibling breeding but the only way to do it.

Question 24

When you have produced the phenotype in forward genetic screen you want to identify the mutated gene. What are the three ways you can do this?

Answer 24

• By recombination (classical method).
• By complementation.
• By whole-genome sequencing.

Question 25

How does mapping by recombination work? (not really used anymore since sequencing is so accessible now, but good to know!)

Answer 25

Map the gene to chromosome/smaller region by crossing with strains carrying genetic markers (like known recessive mutations giving visible phenotypes or sequence-based, e.g. single nucleotide polymorphisms, SNPs). If those known traits come up, you can use the frequency of that to tell if they're on the same/different chromosome. The shorter the distance between a marker and the mutation causing your phenotype, the more difficult it will be to obtain a strain with both of these alleles (requires recombination). When having obtained a rough location of the gene, use complementation (sequence based mapping).

Question 26

How does complementation mapping work?

Answer 26

After recombination mapping, complementation mapping was traditionally used (before sequencing was readily available) to tell whether the phenotype were due to different alleles of different genes. Complementation mapping is a way to identify multiple alleles of the same gene. Cross mutants with each other or inject fragments of the chromosome in mutant germline, and if the progeny got the WT phenotype = complementation. If progeny still had mutant phenotype = no complementation. All mutants within a complementation group fail to complement each other because they have different mutant alleles of the same gene.

Question 27

What is meant by out-crossing in forward genetic screens?

Answer 27

Number of background mutations can be reduced by crossing mutant to wild type/starting strain = back-crossing or outcrossing. Very useful to narrow down the number of mutations.

Question 28

How do we identify the mutated gene today?

Answer 28

Mostly by DNA sequencing. Whole genome resequencing to identify mutation causing the phenotype! No (or only little) genetic mapping required. Feasibility depends on size of genome (but not much longer).

Question 29

What is an epistasis analysis used for and how does it work?

Answer 29

An allele of one gene that masks the phenotype of another gene is said to be epistatic to the other. And when doing an epistasis analysis, you construct double mutants to tell whether two genes act on the same pathway. For example, you construct mutants with two different alleles (eg red hair and blond hair) and with another gene that you suspect has epistasis over the hair color gene (eg baldness) --> look at mutants, since only bald phenotypes appear, the bald gene has epistasis over hair color gene.

Question 30

When doing forward genetics to identify genetic diseases in humans, we obviously can't produce progeny and interbreed them lol. How do we perform forward genetics on human genetic diseases today?

Answer 30

We analyze cohorts of people affected/not affected to find markers linked to the disease, often by looking at single nucleotide polymorphism sites through SNP arrays (to find differences in places where we expect people to differ and find common denominator in affected group compared to not-affected group. We can also do whole genome resequencing but that is not widely used yet bc expensive and a loooot of data to go through.

Question 31

What is reverse genetics used for?

Answer 31

Studying gene function. This is accomplished by silencing/overexpressing genes or destroying gene products (or other changes) and seeing what phenotype is produced.

Question 32

In reverse genetics you perform gene alteration/inactivation/overexpression to produce a phenotype. The most common is gene inactivation and there are several methods for this, but they all fall in one of two classes, which?

Answer 32

- knockout = removal of gene (or part of gene, creating null allele) in DNA, eg by CRISPR-cas) base editing. - knockdown = (temporary) decrease in gene expression, no change in DNA sequence. Eg. RNAi The selection of method depends on which organism you are studying!

Question 33

When doing reverse genetics we have a prediction that something will change when altering the gene, but what do we need to think about to move further?

Answer 33

- On which level? RNA, protein, post-translational, metabolic, something else? - we can either make an educated guess/hypothesis on how things will change, eg based on BLAST search to see what phenotypes can be produced by similar changes. - If we can make a very educated guess, a low throughput method can be enough, but if we need to look globally in the organism/cell we need HTS methods ("omics").

Question 34

Explain the terms "transcriptomics", "proteomics" and "metabolomics".

Answer 34

Transcriptomics = study of gene expression on RNA level Proteomics = study of gene expression on protein level metabolomics = study of metabolic output

Question 35

How does RNAi work in short?

Answer 35

1. To perform RNAi we produce dsRNA that is complementary to the mRNA of the gene we want to silence and inject it into the organism/cell. 2. The dsRNA is recognized by a protein called dicer which fragments it into siRNA (small interfering RNA) 3. The siRNA bind to a protein in the argonaut family which promotes the formation of the immature RNA induced silencing complex (RISC) 4. RISC matures by ejecting the sense-strand of the siRNA and this changes its conformation to an active state. 5. The antisense siRNA strand in the active RISC complex bind to the complementary mRNA sequence and cleaves it. 6. The cleaved mRNA is quickly degraded and this results in (temporary) silencing of the gene producing it.

Question 36

What is the function of RNAi in cells?

Answer 36

RNAi pathways are present in most eukaryotes, but has been lost in e.g. budding yeast. It probably started as a defense against viruses and transposable elements, as a way to defend against non self. But RNAi also regulates endogenous genes and is an essential mechanism for fine-tuned control of translation in eukaryotes. Eukaryotic mRNA is more stable than bacterial mRNA, and because degradation of some mRNAs is stochastic, cells must be able to tightly control which mRNAs will be translated into protein. During development, it is especially critical to ensure rapid and complete turnover of key mRNAs.

Question 37

What three methods can be used for RNAi delivery in C. elegans?

Answer 37

Injection, soaking or feeding. RNAi is very potent in moving over membranes, so any route of administration will result in RNAi in the whole organism.

Question 38

Can any form of dsRNA be used for RNAi in eukaryotes with RNAi machinery?

Answer 38

No, mammals will recognize long dsRNA (>100bp) as viruses and degrade it, so it can't be used for RNAi. In mammals dsRNA must be siRNA (21bp) or shRNA (short hairpin RNA) to work. In other eukaryotes long dsRNA works fine.

Question 39

Two things need to be especially considered to ensure efficient RNAi, which and why?

Answer 39

- off target effects: The siRNAs can be complementary to other parts of the genome. - silencing efficiency: make sure the siRNA isn't too complementary to itself.

Question 40

What three methods can be used for RNAi delivery in mammalian cells?

Answer 40

- lipid nanoparticles with receptors on the outside thar bind to target cells to facilitate endocytosis. - siRNA conjugated to sugar (most common): essentially highjacking a transportation route that already exists (as sugar is actively transported into cells), or cholesterol to be taken up selectively by liver cells. - Viral vector: placing a vector of dsRNA in deactivated virus which transports plasmid into nucleus via the regular viral infection route. In nucleus its opened and transported out into cytoplasm.

Question 41

What are the biggest differences between forward genetic screens and RNAi screens (reverse genetics)?

Answer 41

In forward genetic screens we can produce any kind of mutation - any gene and several types of mutations possible (LoF, GoF, tissue specific genes etc) while not all genes are susceptible to RNAi and we an only reduce expression, no other types of mutation. In forward genetics mutations are confined to one gene and mutant alleles are heritable, while in RNAi multiple genes can be knocked down and itäs usually not heritable.

Question 42

Homologous recombination was used for gene knockouts primarily before CRISPR/cas9 was discovered, today still used in budding yeast and mice. How does it work?

Answer 42

Homologous recombination utilizes the DNA repair machinery already present in cells to delete, insert or alter specific genes by introducing foreign DNA into the genome. 1. use homologous sequences on the flanks of primers and amplify new gene, which creates DNA with new gene in the middle with flanks homologous to DNA in target cell. 2. introduce amplified DNA into cell and hope that it is inserted into genome instead of the DNA where the old gene were. (can also result in donor DNA being inserted elsewhere) 3. Knockout achieved! Note: effective in budding yeast but only about 1/1000 target replacements in mammals. Solution: positive/negative selection. Not effective in Arabidopsis/C. elegans.

Question 43

How did we initially solve the problem of having such low insertion rate of homologous recombination?

Answer 43

By introducing DSBs, which increased the rate of HR a lot! But one problem remained, to induce the DSBs in the target spot. Solved with CRISPR/cas9!

Question 44

Homologous recombination (HR) gene replacement works quite well in mice, how is it done?

Answer 44

1. Introduce amplified DNA with homologous sequence flanks into embryonic stem (ES) cells and hope that it is inserted into genome instead of the DNA where the old gene were. 2. Grow cells on plate to form colonies and select for ES cells with insertion 3. Mate mice and wait three days, isolate early embryo. 4. Inject ES cells with insertion into early embryo. 5. Introduce embryo into pseudopregnant mouse. 6. test somatic cells of offspring partially formed from ES cells with gene insertion for the presence of it. 7. breed positive mice together to produce strain of transgenic mice with altered gene copy in germline. A long and very inefficient method, so CRISPR-cas9 was revolutionary for this!

Question 45

What does CRISPR stand for?

Answer 45

Clustered Regularly Interspaced Short Palindromic repeats

Question 46

What is the natural function of CRISPR/cas systems?

Answer 46

CRISPR/Cas systems are adaptive immune system in bacteria and archaea that protects against phage infection by specifically targeting phage DNA (previously encountered). When identifying previously encountered phages, the CRISPR/cas machinery make DSBs in the sequence it has saved from previous infection of that phage, which kills it very efficiently.

Question 47

How does CRISPR/cas9 work in short?

Answer 47

- A chimeric single-guide RNA (sgRNA) consisting of a CRISPR RNA (crRNA) with a complementary sequence to target DNA and trans-acting crRNA following crRNA containing a sequence that binds the endonuclease cas9, binds to cas9 and target. - The target genomic loci needs to have a Protospacer Adjacent Motif (PAM) which is a NGG sequence in DNA, this is not present in the bacterial genome to make sure it doesn't cut its own genome. - Cas9 cleaves the target DNA when everything is aligned, resulting in a DSB at the target, which can be anywhere in the target genome. - To enable insertion of donor DNA, the flanks of the donor DNA need to be homologous to the target DNA, and homology-directed repair (HDR) leads to insertion of the donor DNA at the target site. Another possibility is that the non-homologous end joining (NHEJ) machinery (quick and dirty) cause indels (insertion/deletion) at the target instead. These two mechanisms complete so target insertions are not 100%.

Question 48

The CRISPR/cas systems have a lot of applications, give three examples of use cases.

Answer 48

- In animal/plant breeding: to introduce genes for example drought tolerance in plants or resistance to a virus. - Medical applications: removing antibiotic resistance in bacteria or gene therapy. - Gene silencing (CRISPR/cas base editing) - creating transgenic organisms for scientific purposes and soooo much more!

Question 49

What are the major differences in gene editing in somatic vs germ cells?

Answer 49

The biggest difference is that somatic cell gene edits are only local, many cells need to be modified to see an effect and edits can't be inherited. With germ line editing you only need to edit one cell and the changes will be permanent in all cells originating from that cells, so it's inherited in progeny. Germ cell editing is illegal in most countries because of the uncertainty of what changes can bring.

Question 50

A common problem with the "classical" CRISPR/cas9 approach is unwanted indels and off target effects, what new approach have been developed to minimize these problems?

Answer 50

- CRISPR base editing: Which doesn't produce DSBs and therefore minimizes the risk of indels. CRISPR base editing works by using deactivated Cas9 (dCas9) that doesn't have endonuclease activity, but instead is fused to a protein that modifies a base, for example CRISPR adenine base editors that deaminate adenine which leads to the formation of inosine which is interpreted by DNA polymerase as guanine, leading to a substitution of A→G. - CRISPRi/CRISPRa – dCas9 fused to transcriptional repressor/activator that activates/represses genes. There's also non Cas9 systems (e.g Cpf1/Cas12a, Cas13) with other properties.