Genetic Engineering

what is the general approach to forward genetics

1. mutagenise germline of WT organism

2. self-fertilise or undertake crosses to produce homozygotes for mutation

3. screen for phenotypic changes of interest

4. map/sequence to identify impacted gene

5. use experiments to determine molecular role/interaction of impacted gene

what is transposon mutagenesis

utilizing transposon and restriction enzymes to insert a transposon into germline cells

• tagged so you can observe this

what are major advantages to chemical/X-ray mutagenesis

• mutants can be labelled/selected for

• inserted gene easily identified

what are some pros/cons to forward genetics

PROS:

• genome-wide

• unbiased

CONS:

• mapping/screening for impacted gene can be difficult

• unfocused (you might be interested in particular genes)

what is reverse genetics

starts with a known DNA sequence and manipulates it to understand the gene's function/phenotype

• mutant gene to alter function

• modify expression

• introduce gene to another organism

what needs to be introduced so gene expression/structure can be altered

nucleic acids involved in the system, introduction may be:

• transient: exists within/for limited number of generations

• permanent: integration with cell’s DNA via recombination

what is the RNAi pathway (summary)

RNAi = gene silencing

1. Dicer cuts dsRNA

2. small RNAs load into RISC

3. guide strand directs RISC to matching mRNA

4. mRNA is cut or blocked

5. protein not made.

how can the RNAi pathway be used for gene knockdown

we insert the dsRNA that participates in the pathway

• transient: dsRNA introduced to cells/organism

• permanently: dsRNA ‘gene’ introduced as transgene

what are some pros/cons for RNAi knockdown

PROS:

• provides control via administration

• can target all genes in genome

• works in many eukaryotes

• variable knockdown efficiency

CONS:

• variable knockdown efficiency

• off-target effects

• not applicable in all eukaryotes

how is homologous recombination used for gene knockouts

homologous recombination can be used to “swap” a functional copy of the gene out for a “knockout” copy of the gene

how can positive & negative selection avoid random integration

when homologous recombination occurs, we can have 2 outcomes:

• double crossover OR one crossover

since we want a double crossover we can insert a negative marker, this marker will help us find sample that have only undergone one crossover, so they can be removed

what is a knock-in gene

introduce a novel gene function/expression

• entirely new gene

• novel component of existing gene

• change to a nucleotide

what are some pros & cons of homologous recombination

PROS:

• directly target genes

• highly efficient

• relatively cheap

CONS:

• based on occurrence of random events

• depends of rate of homologous recombination

• off target effects

summarise the process of CRISPR in bacteria

• Spacer acquisition: Cas1–Cas2 grab a short DNA “mugshot” (spacer) from an invader and insert it into the CRISPR array.

• Expression: The array is transcribed to a long pre-crRNA, then cut into individual crRNAs that each carry one spacer.

• Interference: A crRNA loads into a Cas effector (e.g., Cas9 or Cascade). Guided by sequence matching and a nearby PAM, it finds the invader DNA/RNA and cuts it.

what are some pros & cons of CRISPR-Cas9

PROS:

• efficiently target any gene

• quick & relatively cheap

• works in all organisms tested

CONS:

• intended alteration may not be achieved

• cleavage displays infidelity (off target effects)

what is the GAL4/UAS system

a system that drives gene expression of a transcript of interest

• placing GAL4 expression under a promoter makes this conditional

• promoter can be cell/tissue specific

what are some ways that GAL4/UAS system can be controlled

• controlled via temperature

• controlled via inducer

what is the Cre-lox system

a system that conditionally excises genetic material based on the expression of a recombinase

• system can be inducible by an agonist