Reverse genetics Flashcards
What is reverse genetics as opposed to forward genetics?
Reverse genetics: starting with a known gene and finding out the phenotype it causes by manipulating the gene (overexpression/repression, knockout etc) –> change in phenotype?
Forward genetics: starting with a known phenotype and tracking down the gene(s) that causes the phenotype.
List the two most common methods for gene knockdown in reverse genetics?
RNAi
CRISPRi (CRISPRa and CRISPRi)
List the two most common methods for gene knockout in reverse genetics?
Homologous recombination - for example Lambda red.
CRISPR-Cas9
Why are long dsRNA more rarely used in RNAi in mammals? What is used instead?
long dsRNAs often have a non-specific response in mammals, which may lead to cell death. Instead, shRNA or siRNAs are used.
What is the general concept of RNAi?
RNAi is used for gene knockdown (mRNA silencing) and involves injection/expression of a dsRNA that will degrade the target mRNA temporarily.
What is RISC and what does it stand for?
RISC = RNA induced silencing complex. The protein complex involved in RNAi. The dsRNA that is separated into single strands guides the RISC to the target mRNA for degradation.
What are the four main methods of RNAi delivery in C. elegans?
FIST
Feeding: Expressing the dsRNA in bacteria and feeding to the worms.
Injection: Expressing dsRNA in vitro and injecting into worms.
Soaking: Expressing dsRNA in vitro and soaking the worms in it.
Transgenic expression: inserting the gene into C. elegans and expressing transcript in vivo.
What are the three main methods of RNAi delivery in mammals?
siRNA via lipid nanoparticle: the nanoparticle is covered in antigens which binds to a protein on the membrane –> endocytosis into the cell.
siRNA conjugated to sugar: siRNA is taken up by cell via a glucose transporter and endocytosis.
Viral vector: The viral vector carries DNA template for the siRNA –> transcribed to shRNA and diced to siRNA.
How does gene knockout by PCR work?
When amplifying the fragment you want to insert with PCR (for example a resistance cassette), sequences that are homologous to the flanks of the target gene are added to the primers. The product is then introduced to target cell via transformation, and hopefully the fragment will recombine with and replace the gene to delete.
What is one method to increase the efficiency/probability of homologous recombination at the target site when introducing an insert?
Double stranded breaks (DSB). DSBs increase the risk for mutations and therefore have to be repaired fast. By introducing both a DSB at the target site and an homologous insert, the efficiency of HR is increased.
How is CRISPR-Cas9 used in the context of gene knockout?
The CRISPR system is used to introduce DSBs at the target site. CRISPR increases the specificity of the insert more than just by using homologous recombination. A guide RNA is designed to target the specific site and transcribed together with tracrRNA –> they guide the Cas9 protein to introduce a DSB at the site –> introduce the insert with homologous flanks.
One major problem with CRISPR is that unwanted off-target effects and InDels occur (especially a problem when used in humans). What are two methods to reduce these effects?
Prime editing: development of CRISPR editing that introduces single stranded breaks instead of DSB - decreases the risk of off target effects.
dCas9: usage of an inactivated Cas9 - does not cleave the target but can fuse to target sites and proteins in order to enhance or inhibit expression.
How does the process of using CRISPR for gene editing in for example a mouse?
Mouse gametes are fused to generate a zygote, which is taken out. A sgRNA, Cas9 and a repair tempate (the sequence you want to insert) is injected into the zygote in vitro, whereupon the zygote is transferred into a surrogate mother mouse. The offspring will hopefully carry the CRISPR changes.
What is the main reason that CRISPR gene editing can be more beneficial than using traditional ESC targeting?
CRISPR is more time-efficient and is not limited to only one species (like in ESC where only mice are used). The reason CRISPR is more time efficient is that the gene editing is performed in the zygote genome, allowing the changes to occur already in the first offspring generation. In ESC, the edited gene is injected into embryonic stem cells that are grown into a culture and then injected into the blastocyst. The offspring therefore become chimeric, and several generations are bred to obtain individuals with the full change.
