- Outline the process of molecular cloning of a gene or segment of DNA
- Describe appropriate methods for cloning eukaryotic genes so their protein products can be expressed (transcribed into mRNA, which is then translated to make protein).
- Compare and contrast the vectors and procedures used for creating genetically modified bacteria and eukaryotes
- Describe genome editing with CRISPR-Cas9 – what is required for targeting and how target genes can be modified
Many of our drugs, much of our food, and even our clothing are now produced using recombinant DNA technology. Instead of depending on random mutation, and either natural or artificial selection, we now have the ability to directly manipulate the genes of organisms to create new proteins and new capabilities in our domesticated bacteria, fungi, plants and animals.
Molecular biologists coined the term “molecular cloning” to describe the process of selectively replicating a chosen segment of DNA. The cloned DNA segment may be replicated within a cell, using “recombinant DNA” technology, or in a test tube, using the polymerase chain reaction (PCR).
Recombinant DNA technology leads to genetically modified organisms (GMOs). Recombinant DNA requires 3 key molecular tools:
- Cutting DNA at specific sites – most often performed by enzymes called restriction endonucleases (restriction enzymes). Restriction enzymes often make staggered cuts at specific 4, 6, or 8-bp palindromic sequences in duplex DNA, leaving characteristic “sticky ends” that can anneal to each other via hydrogen bonding between complementary bases on the single-stranded overhangs.
- Ligating DNA fragments with an enzyme called DNA ligase. DNA ligase, the same enzyme used during cellular DNA replication to knit together Okazaki fragments, creates covalent phosphodiester bonds between any two DNA fragments that have been cut by the same restriction enzyme, or have the same compatible “sticky ends”.
- A “vector”, such as a plasmid, that can be used to insert a new segment of DNA via restriction enzyme cutting and ligation. The plasmid containing the inserted DNA segment will replicate in host cells.
Also see: https://www.dnalc.org/resources/3d/20-mechanism-of-recombination.html
The alternative to using recombinant plasmids is to directly copy and amplify a specific DNA segment using PCR. PCR requires a pair of primers that correspond to the ends of the desired DNA segment.
Even random DNA segments, where the base sequences are unknown, may be amplified by ligating adapter primers, short synthetic DNA segments of known sequence, to the ends of the target DNA molecules.
Cloning eukaryotic genes
Molecular cloning of eukaryotic genes is often either unfeasible or undesirable, or both, because they contain numerous, large introns. Plasmid vectors have a practical size limit of less than 10 kilo-base pairs (kbp), and PCR is also difficult beyond about 10 kb.
The mRNA, lacking introns, is a compact version of a eukaryotic gene that retains all of the protein coding information. The enzyme reverse transcriptase can be used, along with an oligo-dT primer that is complementary to the polyA tail, to synthesize a complementary DNA (cDNA) molecule. The cDNA can then be cloned into a plasmid or amplified by PCR by ligating adapters that contain restriction endonuclease cleavage sites or PCR primer sequences.
Expressing cloned genes: genetically modified organisms
A genetically modified organism (GMO) is any organism that has been manipulated to carry new genetic material, from either a different species or synthesized in the laboratory. The point of creating GMOs is usually to alter one or more traits in the modified organism, most often so they express a new gene. Examples include pathogen resistance or toxin expression to reduce herbivory.
Expression of foreign genes in bacteria
Plasmid vectors for cloning and expression in bacteria (see pUC18 map above) must have
- An origin of DNA replication (ori) that directs their replication in the host cell
- restriction endonuclease sites (“polylinker”) that occur just once on the vector and allow insertion of cloned DNA segments
- a selectable marker gene, such as antibiotic resistance (bla encodes beta-lactamase for ampicillin resistance), so cells that do not contain the plasmid can be eliminated
- a way to distinguish cells that have the original plasmid from cells that have a recombinant plasmid.
- a promoter to drive transcription (and translation) of the inserted foreign gene
The last feature is important because the ligation of plasmid and foreign DNA segments favors the plasmid ends re-ligating without a foreign DNA insert, resulting in the original, “empty” plasmid with no foreign DNA insert. Plasmid vectors therefore have the cloning site within a second antibiotic resistance gene or within the lacZ gene (encodes beta-galactosidase). Insertion of a foreign DNA segment will disrupt the gene. Colonies of E. coli cells that have empty plasmids (no inserted foreign DNA) have an intact lacZ gene, produce functional beta-galactosidase, and cleave a colorless dye called X-gal to release the insoluble blue dye X, and turn blue. E. coli cells that have plasmids with foreign DNA inserts make no beta-galactosidase, and are unable to cleave X-gal. These colonies stay white. Blue colonies are discarded, and white colonies are picked for further testing.
Cloning into the 5′ end of the lacZ gene also means that the E. coli cell can express a protein encoded by the inserted DNA. The lac promoter provides a means to regulate transcription, and protein coding sequences in the inserted DNA can be expressed as a fusion protein, containing the first few amino acids of the E. coli beta-galactosidase gene, and any amino acids encoded in the same reading frame by the inserted DNA sequence.
Expression of foreign genes in eukaryotes
Vectors for expression of foreign genes in eukaryotic cells must provide appropriate eukaryotic promoters upstream of the cloning site, for transcription by the eukaryotic host cell, as well as downstream polyadenylation and transcription termination signals. For single-celled organisms such as yeast, and cultured cells, bacterial plasmids containing foreign genes can be transformed into the cells. The plasmid DNA gets into the nucleus, and inserts into random locations in the host cell’s chromosomes. For multicellular organisms, the delivery of genes into the cells of the organism poses special challenges and requires special vectors and delivery methods. We describe these challenges for one application, human gene therapy, in the next section.
Gene therapy poses a special challenge in delivering recombinant DNA into host cells. Recombinant DNA technology can readily clone a functional copy of a defective gene and insert it into a vector with the correct regulatory sequences. But how can we deliver this functional gene into the cells of a person who has already been born? The most promising techniques use viruses. Viruses evolved to be highly efficient at delivering their own genetic information into host cells. Replacing the viral replication genes with a therapeutic human gene eliminates the ability of the virus to replicate, while co-opting the viral infection mechanism to deliver the therapeutic gene into the nuclei of host cells.
Even then, only a small percentage of cells are infected and repaired (remember these therapeutic viruses can’t replicate to infect other cells). Moreover, benefits of viral gene therapy are short-lived, as the “repaired” cells age, die and are replaced by genetically unmodified cells.
A promising solution to these challenges is to find and genetically modify stem cells, those cells that will continue to divide and replenish the body’s cells for the rest of the patient’s life. Genetically modified stem cells can be returned to the patient’s body and have the potential to supply and replenish genetically modified blood cells and tissues for the rest of the patient’s life.
One technology developed in recent years, and being widely adopted in research labs around the world, is CRISPR-Cas9 technology, and variants. CRISPR stands for Clustered Regularly Interspersed Short Palindromic Repeats and functions as a library of foreign DNA samples that represent viruses or pathogens that could harm a bacterial species. Cas9 is a protein enzyme that binds short RNAs made from the CRISPR gene library. Cas9 bound to a short RNA can recognize corresponding DNA sequences that match the CRISPR RNAs, after which Cas9 will cleave or cut the DNA, causing a DNA break. This discovery of CRISPR-Cas9 from bacteria generated a molecular technology that enables researchers to delete, add, or replace particular bits of DNA in a cell.
Here is a TED talk video by Jennifer Doudna, one of the developers of CRISPR technology and a winner of the 2020 Nobel Prize for Chemistry:
In essence, Cas9 is a protein that cuts DNA, which causes a DNA break. Whereas restriction endonucleases cut DNA at fixed sites, Cas9 is programmable. Cas9 targets the DNA site to be cut by using a short guide RNA (sgRNA). Cas9 binds the sgRNA, and cuts DNA wherever the sgRNA binds to a complementary DNA sequence. So, in any organism where the genome sequence is known, scientists can make an sgRNA to target a particular DNA sequence in the genome, and cut it. After Cas9 cuts the DNA to create a double-strand DNA break, the cell’s DNA repair system will trim the broken ends and ligate them together in a process called non-homologous end-joining, which often creates a small deletion as a result of the trimming. If instead a homologous DNA sequence that matches the sequences around the cut ends is available, the cell’s DNA repair system can use the matching DNA as a template to repair the break in the DNA. This homology-dependent repair system loops in the sequence information in the homologous DNA template to repair the broken DNA. By providing Cas9 protein, sgRNA, and a homologous template DNA that includes a desired change, scientists have successfully made precise changes in genomes of many kinds of cells and organisms, including cultured human cells.
As a society, we are currently debating whether human genome editing is less controversial than human genetic modification. In CRISPR-Cas9 gene editing, no non-human DNA need be added. Want to know more? The ethical discussions around gene editing in humans are especially contentious surrounding implanted embryos. The overwhelming majority of researchers believe the technology is not yet precise or accurate enough to attempt edits in human embryos in vivo. However, in 2018, Chinese biophysicist He Jiankui released claims of editing three human embryos that were then implanted and brought to term as live births. As a consequence, Chinese authorities reported He went to prison for three years.
Put it all together:
In class we will discuss how these concepts are applied to current gene therapy methods undergoing research and development.
Dr. Choi’s lecture video on recombinant DNA technology (in one 39-min chunk, until I find time to redo this in multiple short segments):
I believe the molecular cloning 3 steps for recombination DNA tech. are out of order. Isn’t Step 3 and step 2 supposed to be switched?
I see your point. I edited to change the word “steps” to “operations” to lessen any sense that these are an ordered sequence. The order of operations would be: first, cut both the target DNA and the vector DNA with restriction endonucleases; second, ligate the target DNA fragments with vector DNA; third, transform bacterial cells with the products of the ligation reaction (if the vector is a plasmid). Thanks for pointing out a potential source of confusion.
A good video explaining CRISPR/Cas technology:
November 2016 – added section on genome editing with CRISPR-Cas9. Modified 2nd and 3rd LOs to be more specific, replaced plasmid cloning figure with new figure that correctly shows sticky ends (using EcoRI as the exemplar restriction enzyme), and two possible products of ligation with foreign DNA fragments.
A new paper on inhibitors of Cas9, predicted as an evolutionary response by bacteriophage to inhibit cleavage of their genomes by Cas9: http://www.cell.com/cell/abstract/S0092-8674(16)31589-6