GEN 9: Engineering the Genome Flashcards
1
Q
Observe the learning outcomes for this session
A
2
Q
What happens during natural bacterial transformation?
What is it?
A
- this is the uptake of extracellular DNA that can occur under natural bacterial growth conditions
- some bacteria possess surface proteins that can transport DNA to closely related cells allowing a process called transformation
- when a bacteria cell dies, it can break open and release DNA which can be taken in by closely related species and incorporated into their genomes
3
Q
Observe the diagram and describe in more detail the pathway components of bacterial transformation
A
- transforming DNA:
- extracellular DNA released in the environment (e.g. by dead bacteria) can bind non-covalently to sites present on the cell surface
- competence-specific single-stranded DNA binding protein:
- protein that binds to ssDNA and is only expressed in bacteria that are naturally competent
- uptake of ssDNA:
- double-standed DNA is converted to ssDNA, which is taken into the cytoplasm
- homologous recombination:
- extracellular DNA is incorporated into the bacterial genome by homologous recombination (HR)
- the transforming DNA must be homologous to the recipient bacterial genome
- i.e. closely related bacterium
4
Q
Describe what competent bacteria are
A
- competent bacteria are capable of taking up DNA
- some bacteria are naturally competent
- e.g. some members of the genera Campylobacter, Staphylococcus and Streptococcus
- others are not
- e.g E. coli
- Non-competent bacteria can be made artificially competent.
5
Q
In what form of DNA do naturally competent bacteria take up exogenous DNA?
Why is this?
A
- Naturally competent bacteria take up exogenous DNA as linear fragments that they convert into ssDNA
- they do not take up circular plasmids and must be made artificially competent to do this.
- natural competence is a biological state that requires expression of certain host cell genes
6
Q
How does artificial competence occur?
A
- artificial competence is a temporary state induced by chemical (salts) and physical (heat-shock) treatments that allow DNA to enter the cell (including circular, double-stranded plasmid DNA).
7
Q
Linear single-stranded DNA is converted to double-stranded DNA during DNA uptake in natural bacterial transformation.
True or False?
A
- false
8
Q
Integration of naturally transformed DNA in the bacterial chromosome occurs by homologous recombination.
A
- true
9
Q
Bacterial transformation is the natural mechanism by which plasmid-borne antibiotic resistance spreads.
A
- true
10
Q
Artificially competent bacteria are used in the laboratory to take up circular plasmids.
A
- true
11
Q
Why is it advantageous for bacteria to have a system for recombining DNA into their genomes?
A
- because natural transformation increases genetic diversity
- Bacteria reproduce asexually by binary fission, a process which generates two genetically identical progeny cells (apart from rare random mutations).
- The ability of organisms to adapt to environmental changes requires a genetically diverse population, where the individuals best suited to the environment will survive and proliferate.
- Natural transformation enhances bacterial genetic diversity by transferring sections of DNA from one individual to another: it ‘shuffles the pack’ of available variant genes in the population, rather like meiosis does in eukaryotes.
12
Q
What are selectable marker genes and what did they allow?
A
- selectable marker genes allowed gene targeting so that it was possible to modify genomic loci of unknown function
- allowing for reverse genetic approaches
- an example of a gene could be genes that confer resistance to an antibiotic
- these approaches were developed in bacteria as well as in yeasts, which can also incorporate exogenous DNA into their genomes by HR
13
Q
Explain this simple example of replacement construct gene targeting
A
- Here, the replacement construct is a linearised plasmid made in the laboratory. It carries regions of homology with the chromosome (b and c), separated by a marker gene (red bar). Regions of the plasmid which are not necessary for this type of gene targeting (dashed blue line) are ideally removed.
- After delivery into cells, the construct aligns with its homologous regions in the chromosome (dotted black lines), and undergoes HR involving two crossovers (large Xs).
- The resulting replacement event introduces the marker gene between regions b and c in the chromosome. Cells modified in this way are selected in antibiotic. If regions b and c are known to carry a gene, the effect on cells of disrupting it in this way can be studied.
14
Q
Describe gene targeting with an insertion construct
Why is it used?
A
- It is often desirable to make a more subtle chromosomal modification such as a single nucleotide change
- Step 1: A single crossover (X) between one homologous region (b) results in insertion of the entire plasmid into the target locus, and duplicate copies of the homology region (b and c). Cells that have undergone this event will express the antibiotic resistance gene (red bar).
- Step 2: A second crossover (X) between one homologous region (c) on the other side of the mutation (red asterisk) results in excision of the insertion construct, which now lacks the mutation. The original target locus is left modified only by the subtle mutation.
15
Q
What is transfection?
How is this different from transformation?
A
- these are the methods developed for artificially introducing DNA into mammalian cells
- transformation is not used because in mammalian cells it refers to the acquisition of malignant characteristics.
16
Q
What are some common transfection methods?
A
- viral transfection:
- DNA is packaged into viral particles that deliver DNA into target cells
- advantage: efficient delivery
- disadvantage: time-consuming to make particles
- microinjection:
- direct injection of DNA into the nucleus using a micro-needle
- advantage: efficient
- disadvantage: needs much time and skill
- electroporation:
- a capacitor is discharged through mixed cells and DNA inducing transient membrane pores for DNA uptake
- advantage: useful for large cell numbers
- disadvantage: require special equipment
- lipofection:
- complexes between cationic lipids and negatively-charged DNA are endocytosed by cells
- advantage: simple
- disadvantage: less useful for large cell numbers
17
Q
What happens to DNA artificially delivered to mammalian cells?
A
- Unlike bacteria and yeast, mammalian cells integrate most exogenous DNA into chromosomes at more or less random genomic sites, with no requirement for sequence homology to the recipient genome.
- Initially, this suggested that mammalian somatic cells do not support HR.
- Further experiments revealed that HR-mediated integration does indeed occur in mammalian cells, but at frequencies 10 to 1000-fold lower than random integration.
- Only later was the HR-dependent pathway of DNA double-strand break (DSB) repair in mammalian cells elucidated
18
Q
What is the mechanism that underlies random integration in mammalian cells and why is it preferred over targeted integration?
A
- The mechanism is Non-Homologous End Joining (NHEJ)
- Recall that double-strand breaks occur at more or less random sites throughout the genome and are subject to repair by NHEJ or HR.
- Although NHEJ preferentially re-joins the chromosomal ends at a double strand break, it is error-prone.
- NHEJ errors include not only the formation of chromosomal translocations but also the incorporation of exogenous DNA at double strand breaks.
- Integration of exogenous DNA by HR, by definition, can only occur at the homologous chromosomal site.
- This, combined with the fact that NHEJ is active throughout interphase, whereas HR is inactive during G1, helps to explain the high ratio of random to targeted integration of targeting constructs.
19
Q
Describe the different fates of transfected DNA inside the mammalian cell
A
- Depending on the transfection method, DNA prepared in vitro (1) reaches the nucleus either directly (2) or via the cytoplasm (3)
- Some of the DNA may be degraded during delivery, either in the cytoplasm (4) or the nucleus (5)
- DNA that reaches the nucleus intact may be transcribed into mRNA (6), which is then translated into protein (7)
- such expression is only transient, however, because the DNA will eventually be degraded or diluted as the host cells proliferate
- for its stable/long-term expression, the transfected DNA must integrate into the host genome so that it is replicated as the host cells divide
- integration is most likely to occur at chromosomal DSBs using the NHEJ pathway of DSB repair (8)
- if the transfected DNA is homologous to a chromosomal sequence (9), it may integrate at this locus by the HR pathway of DSB repair (10)
20
Q
Describe random and targeted integration of a replacement construct in mammalian cells
A
- If the transfected DNA is a targeting construct, including a marker gene that confers resistance to an antibiotic, then stably transfected cell clones can be selected in culture medium containing antibiotic.
- Each clone will have at least one copy of the targeting construct in their genome.
- In most clones the construct will have integrated at a random site.
- Only occasional clones will have integrated the construct by HR at its target site.
- These two types of integration, random and targeted integration, are illustrated below for a replacement type targeting construct.
21
Q
Can you think of ways to select, or screen for, rare targeted integrants among a majority of clones that are random integrants?
A
- to be updated
22
Q
Explain the use of a reporter gene to stably transfect mammalian cell lines
A
- Stably transfected mammalian cell lines are important for both research and biotechnology.
- In one research application, a reporter gene, such as the green fluorescent protein (GFP) gene, is linked to cis-acting transcriptional control elements from a gene induced by a signalling pathway of interest.
- After stable integration of the expression plasmid into a suitable cell line, the effects of various molecules or growth conditions on the signalling pathway can be conveniently assessed by measuring the amount of green fluorescence.
23
Q
What are stably transfected cell lines used for in commerical production?
A
- it is used for the commercial production of pharmaceutically important proteins
- e.g. blood clotting factors and antibodies
24
Q
Why are mammalian cell lines used for producing pharmaceutically important proteins?
What else is needed?
A
- Use of mammalian cell lines (preferably human), as opposed to bacteria or yeast, has the advantage that proteins are made with the appropriate post-translational modifications, such as glycosylation, required for full protein function in patients
- To optimise protein production, the genes for such proteins are usually cloned into a vector next to a strong viral promoter.
25
Q
Does it matter where in the chromosome the transgene is inserted in mammalian cells?
A
- In all these applications, it is often sufficient for the transfected gene (transgene) to be inserted at a random chromosomal site.
- The efficiency of randomly integrated transgene expression varies greatly from site to site, however, due to chromosomal position effects.
- These effects reflect the chromatin status of the integration site (i.e. heterochromatin or euchromatin).
- In practice it is therefore necessary to screen several or many clones before one with appropriate expression level is obtained.
- Alternatively, for reliable and reproducible expression, the transgene can be targeted by HR into a ‘safe harbour’ chromosomal site, i.e. a locus such as a housekeeping gene that is known to be permissive for transgene expression.
26
Q
What are vectors in molecular biology?
A
- vectors are agents used to deliver genetic material into cells that will replicate and/or express it
- Vectors used in the commercial production of proteins are called expression vectors (e.g. a modified plasmid or virus).
- In Lab Pod II, you will generate a modified plasmid vector (containing GFP as a reporter gene) to transfect mammalian cells and obtain an engineered cell line.
35
Q
Describe how gene targeting in mice using ESCs occurs
A
- Targeted ESCs are introduced into a host blastocyst that is then implanted into a foster mother.
- The resulting offspring are chimeric, i.e. some of their cells are derived from the host blastocyst and some, from the modified ESCs.
- If the germ cells of chimaeras are ESC-derived, they can be use for breeding to produce offspring whose every cell is heterozygous for the genetic modification.
- Sibling mating then generates animals homozygous for the genetic modification.
- The whole procedure, as used in mice, is summarised in the following image:
36
Q
What are some of the possible uses of transgenesis with ESCs?
A
- generating knockouts:
- modifications that disrupt or remove a target region (knockouts) are useful for assessing the function of genes or other genomic features
- studying complex body systems:
- transgenic mice are particularly useful for analysing genomic regions controlling complex physiology that cannot be studied in cultures cells (e.g. brain functions)
- studying human disease:
- subtle mutations that mirror human disease-causing mutations have been introduced into mice to generate animal models of human genetic diseases
- e.g. cystic fibrosis mouse
- studying gene expression:
- reporters: such as the GFP gene, can be inserted into a target gene to monitor its expression in live animals
38
Q
Understand how SCNT with target integration is used to generate a pig model of cystic fibrosis (CF)
A
41
Q
Describe the Cre/Lox system
A
- when Cre recombinase is expressed, it finds LoxP sites, cleaves the DNA
- recombines it, excising the portion of DNA in between
- this system can be used in mammalian cells by using gene targeting to introduce LoxP sites around a target gene
- this is performed in a responder animal
- but the Cre recombinase comes from a regulator mouse and is inserted under the control of a specific promoter or inducer
- when the two are mated, the offspring will have both the Cre gene under the specific promoter and the target gene flanked with LoxP sites
- the gene is floxed
- the floxed target gene is knocked out only in specific tissues
48
Q
What stimulates gene targeting by at least 1000-fold?
A
- when an endonuclease is used to cleave the target locus
49
Q
Describe CRISPR
- presence in bacterial genomes
- adaptive immunity
A
- CRISPR stands for clustered regularly interspaced short palindromic repeats.
- These are present in regions of bacterial genomes that store bacteriophage DNA as a marker of prior infections, and they are expressed as natural guide RNA (gRNA) molecules.
- If a repeat bacteriophage infection occurs, an endonuclease (e.g. Cas9 in S. pyogenes) is expressed and guided to the viral genome by the specific gRNA.
- The endonuclease can then cleave the virus and clear the infection.
- This process can be described as a prokaryotic adaptive immune strategy, and it is illustrated in the image below.
50
Q
Describe the three stages of CRISPR-Cas Adaptive immunity
A
- Adaptation:
- During this phase, the bacterium incorporates a fragment of the invading phage or plasmid DNA into its genome as a spacer into the CRISPR array
- leader: thick grey line, repeats: black boxes, spacers: coloured diamonds
- this spacer will serve as memory allowing the bacterium to recognise the same threat upon reinfection
- after infection, the Cas1-Cas2 complex (dark blue rectangles) recognises the invading DNA and integrates a portion of it (orange) into the CRISPR array
- this gives rise to a new spacer (orange diamond)
- at the same time, a new repeat is generated - crRNA biogenesis
- the CRISPR array is transcribed as a long precursor CRISPR RNA (pre-crRNA)
- which is then processed (as indicated by the black arrow) to generate mature CRISPR RNAs (crRNAs)
- each containing part of a repeat and of a spacer
- processing is mediated either by a Cas nuclease (Class 1 systems, types V and VI) or a host factor (type II) - Interference:
- Either a complex of Cas proteins or a singular Cas protein guided by the mature crRNAs cleave the nucleic acid target in a sequence-specific manner (scissors)
- upon base pairing of the crRNA spacer (purple) with the spacer-complementart sequence, knwon as protospacers (purple), a Cas nuclease catalyses cleavage of the invading DNA leading to its degradation
- the PAM sequence, depicted as a green line, is a short sequence found next to the protospacer but absent in the CRISPR array
- which prevents the array to be cleaved by Cas protein (autoimmunity)
- blue rectangle can represent a complex of several Cas Proteins or a single Cas nuclease depending on the CRISPR class
51
Q
How do you perform customised CRISPR gene editing?
A
- requires only the normal Cas9 protein and a gRNA molecule ‘reprogrammed’ to recognise a chosen target sequence.
- The gRNA recognises its target DNA by standard base pairing rules, using just 20 nucleotides (nt) at its 5’ end.
- However, the target sequence is not just any 20 nt, chosen randomly: it must be followed by the sequence NGG (N = any nucleotide), known as the protospacer adjacent motif (PAM).
- The PAM is the DNA sequence that is actually recognised by the Cas9/gRNA complex, but it does not hybridise to the gRNA (see figure below).
- The DNA region with which the gRNA hybridises is known as the protospacer.
- Once bound to its target sequence, the Cas9/gRNA complex cleaves each strand of the DNA, 3 nucleotides upstream of the PAM, using two active sites in the Cas9 protein.
- CRISPR systems from bacteria other than S. pyogenes can be used similarly but recognise different PAM sites.
55
Q
Describe two basic approaches of gene editing using customised nucleases
A
1)
- The nuclease is used without an accompanying DNA (no template): generated DSBs are repaired by error-prone NHEJ, giving rise to indels at the cleavage site.
- Most indels will disrupt gene expression if introduced into coding sequences.
- This approach provides a simpler way to make gene knock-outs than standard gene targeting.
- Furthermore, it works so well in oocytes that multiple target loci can be inactivated simultaneously by injection of multiple nucleases in the same oocyte.
- This approach is also simpler and more efficient than the previous standard route for making knockout mice using ESCs.
2)
- The nuclease is co-delivered with a template DNA (dsDNA or ssDNA) to make a defined modification by HR with the target locus: Frequencies of HR at the target locus are sufficiently high that the use of selectable marker genes is often unnecessary.
- Single-stranded oligonucleotide templates of 60-100 nt can be used to introduce modifications of one or few nucleotides.
- This saves time and expense of making targeting constructs.
59
Q
Label this diagram of CRISPR
A
67
Q
What are some problems and solutions to gene therapy?
A
