Key Technologies Flashcards
What are natural means for introducing genes to cells?
Bacterial transformation - uptake of environmental DNA
Bacterial conjugation - transfer of chromosomal DNA (F factor) between bacteria
Bacteriophage transduction - phage my be lytic, lysogenic or both
What are some artificial means for introducing genes?
Chemical methods - calcium chloride for transformation of E. coli, calcium phosphate for animal cells
Electroporation - produces transient pores, efficient
Biolistics (plants)
Microinjection into egg nuclei
How can we do genetic selection?
DNA can have drug resistance/reporter gene (GFP)
In some cases only small fraction of cells uptake DNA - selected/screened
Introduced DNA can exist extrachromosomally as episome/integrated into chromosomal DNA via recombination
Why are plasmids good vectors?
Direct their own replication and additional factors ensuring the copies get separated into the daughter cells during cell division - not lost during binary fission
Eg. toxin-antitoxin system
How can genes be introduced by transposition?
Transposition is essentially random integration by transposase encoded within transposon
Breaks target DNA and integrates via ends of transposon
Can be replicative/non-replicative
Naturally introduce new genes - vector between inverted terminal repeats
How else are transposons useful?
Can be used as mutagenic agents to disrupt genes and provide a tag to detect it
How does homologous recombination work?
Occurs between regions of homology (quite long)
Involves RecA and RecBCD (E. coli)
Results in integration of incoming DNA, can modify chromosomal sequence
How does non-homologous recombination work?
Doesn’t require homology but can be directed by regions of micro-homology
Major DNA integration method for plants and animals
How can homologous recombination (HR) be used for allele replacement?
Introduced DNA, HR with chromosome/plasmids, Rec-A mediated recombination
2-step method = with plasmid containing homology to genome and +ve and -ve selection markers
Rec-A mediated HR requires pretty large regions of homology
What is Red-mediated homologous recombination?
HR is mediated by RecA pathway and used to make precise modifications - eg introduce gene into BAC - BUT requires large sequences of homology
Alternative = phage lambda Red recombination system - shorter regions of homologous sequence - noe RecA/BC
RecET system from rac prophage similar
Red operon of phage lambda: alpha = endonuclease, beta = annealing protein, and gamma = inhibitor of exonuclease RecBC
How does Red-mediated recombination work?
dsDNA degraded by 5’ to 3’ Red-alpha exonuclease to create ss region
ss coated with Red-beta binding protein
Works by ss annealing, not strand invasion
No need for ATP and only needs short homology arms
DNA target must be replicating
What is Red-mediated ‘recombineering?
Red-mediated recombination exploited for making DNA designed rearrangements in E. coli
Short homologous sequences added by PCR and linear DNA transfected
Alpha, beta, and gamma proteins expressed in E. coli under inducible control
No enzymes required other that PCR polymerase
Rapid and flexible methodfor engineering DNA in E. coli or other bacteria
How do you make linear DNA with homologous ends?
PCR used to make homologous ds linear DNA
3’ ends of PCR primers complimentary to selection marker
5’ ends of primers specify sequence homologous to region in target DNA
Oligonucleotide synthesis allows 5’ ends to be made up to 50-60 nucleotides long for efficient Red-mediated recombination
How does Red-mediated recombineering work?
Lambda Red recombination genes transferred into low copy no. plasmid
Plasmid introduced into E. coli
Linear ‘donor’ DNA made in vitro by PCR and introduced into E. coli
Target DNA incorporates PCR linear and confers drug resistance
How does recombineering with oligonucleotides work?
Recombination with ssDNA (oligonucleotides) very efficient
Bias in efficiency depending on direction of replication fork
Only needs Red-beta annealing protein
Red-beta binds oligonucleotide and anneals it to lagging strand gap in replication fork alongside Okazaki fragments
Can be designed to create diff types of mutation (mismatch, insertion, deletion)
What is MAGE?
Multiplex Automated Genome Engineering
Enables rapid and continuous generation of sequence diversity at many targeted chromosomal locations across a large population of cells through repeated synthetic DNA introduction
Results in rapid evolution of cell population to create strains with desired trait
What is CAGE?
Conjugative Assembly Genome Engineering
Applying conjugation to transfer defined regions of the bacterial chromosome into a recipient strain
oriT derived from F plasmid inserted into desired point on donor chromosome to initiate chromosome transfer; oriT has kanamycin resistance; conjugative functions encoded on plasmid
What is genome editing?
DNA is inserted, replaced, or removed from a genome using artificially engineered endonucleases
What are the two methods of repairing a ds break and when are they most used?
Homologous recombination (HR) - mostly used in bacteria and yeast Non-Homologous End Joining (NHEJ) - mostly used in animal and plant cells
What is a consequence of NHEJ?
Mutagenic as randomly inserts/deletes several base pairs at break site (indels)
How efficient is gene targeting in yeast?
Targeted genome modification occurs 100% of selected cells
Double strand break in the plasmid makes the outcome much more efficient (~100-fold)
How efficient is gene targeting in mammalian cells?
Double strand break also increases frequency of homologous integration but still random integration
What are some problems with DNA injection into egg nuclei?
All integrations occur by non-homologous recombination even if sequence is homologous
Originally could only be used for additive transgenesis
How is plasmid DNA made?
DNA linearised and plasmid backbone sequence removed by restriction enzyme before it’s injected into pronucleus
Contains transgene coding sequence linked to appropriate transcriptional regulatory sequence
How does somatic cell nuclear transfer (SCNT) work?
DNA transfected into primary embryonic fibroblasts and these characterised by DNA analysis to confirm integration state and copy number
Used as donors of nuclei into enucleated eggs
How is gene targeting done in mouse embryonic cells?
Mouse ES cells (derived from pluripotent cells in inner cell mass of embryo) isolated from blastocysts and grown in culture
DNA introduced by electroporation and cells selected with genetic marker
Integration of vector DNA with seq homologous [in mouse ES cells at rare freq
What is the efficiency of targeted gene modification?
~1 in 10^6 in the population of cells
How are targeted gene modifications selected?
Positive-negative selection
DNA of genetically selected cells analysed to confirm correct integration by HR
Cells used for chimera generation and test breeding
Only possible in cell lines like mouse embryonic stem cells that can be easily cultured, proliferate long term, and can be genetically selected by drug resistance
Why are ES cell-derived chimeras important?
Can transmit mutated gene into germ line
Allowed construction of numerous mouse mutants with gene knock-outs
Important for investigations of gene function in development and physiology
Other that rat NOT successfully applied to other species
No applicable to biotechnology industry
How can efficiency be increased for targeted modification of the genome?
Introduce DSB into chromosome rather than plasmid
Cellular repair mechanisms will repair DSB modifying chromosome
What was the 1st gene editing experiment?
ds break in I-SceI endonuclease
Add exogenous DNA template
Homologous recombination
1000-fold increase in gene targeting efficiency
BUT
Strands can also undergo NHEJ - mutagenesis
What are the pros and cons of DSB-induced genome modification?
Pros:
Highly efficient
In principle, possible in any cell/organism
Cons:
Unintended consequences - genetic rearrangements
HR restricted to S and G2 phases of cell cycle; NHEJ throughout
HR and NHEJ in competition
What are alternative options for artificial designed endonucleases with user defined specificity?
Zinc Finger Nucleases (ZFNs) - code for DNA recognition ~3 AA - 3 bp - modify some genes TALE nucleases (TALENs) - ~1 AA - 1 bp - modify any gene RNA directed cleavage: Cas9/guide RNA - watson-crick bp - multiple genes
How do ZFNs work?
Several Zinc Finger Protein units (ZFPs) recognise desired cleavage seq
Fused to FokI cleavage domain - must dimerise to cleave dsDNA, ZFPs require precise positioning
Off target cleavage can occur
How do TALENs work?
TAL effectors = virulence proteins, encode DNA binding domain 33-34 AAs conserved except for RVD which recognises specific nucleotides
Similar design to ZFNs but easier to construct
Off-target cleavage can occur
How does Cas9 work?
Requires guide RNA (20 nucleotides) to recognise target DNA seq and a PAM sequence must be adjacent to target DNA
Endonuclease cleavage based on recognition by RNA-DNA base pairing
How are designed nucleases delivered to cells?
ZFNs, TALENs, Cas9 - expressed from transfected plasmid/from mRNA
Cas9 can also be delivered as a ribonucleoprotein with guide RNA already bound - efficient
How are predesigned targeted mutations carried out?
Need DNA template for homology-directed repair (HDR) needs to be co-introduced
Efficiency of gene targeting so high genetic selection not required often
Mutation can be off-set from position of endonuclease cleavage site
Endonuclease shouldn’t cut the vector or the modified gene after recombination - produced undesired indels by repeated cleavage
How are CRISPR/Cas9 vectors used to generate a mutation?
HDR template designed to introduce mutation AND modify PAM seq (NGG) to prevent further cutting
Variant Cas9 that recognises altered PAM can be used with diff HDR template to restore original PAM leaving only the mutation
What is dead Cas9 (dCas9)?
Has a mutation in both cleavage domains so catalytically inactive but still binds to target DNA seq
Can stop transcription by blocking RNA polymerase
Can bring heterologous effector domain to gene of interest - transcriptional activator, chromatin modifier to modulate gene expression
What is the most flexible genome editing system?
CRISPR-Cas9
Often used in plants in genome editing - legal in America ad not ‘modified’
