Chapter 2: CRISPR and PCR Flashcards
CRISPR regions
- CRISPR is an immune response found naturally in bacteria and is modified in the lab to edit genes
- CRISPR provides a form of NATURAL IMMUNITY for the bacteria against viral attacks
- are DNA with short repeated sequences (repeats) and spacers
- each repeat is separated by a ‘spacer’ DNA
- the spacer DNA is a segment of DNA from bacteriophages the cell has encountered previously
- these clusters are regularly interspaced with the repeat DNA
spacers
- are made up of remnants of DNA from bacteriophages of foreign DNA
- when bacteria encounters a new virus, they store some of its DNA in a new spacer sequence
how does CRISPR-Cas9 work
- a bacteriophage attaches to the outside of bacterial cell and injects its VIRAL DNA into the cell (reinfection)
- previously, a segment of the viral DNA has been stored as a spacer in the CRISPR region
- the CRISPR sequence is TRANSCRIBED resulting in pre-CRISPR RNA (crRNA)
- tracer RNA (trcrRNA) has a complementary sequence to the repeat DNA (NOT the spacers)
- role: helps hold the gRNA in place in the Cas9 enzyme
- RNAase cuts spacer, portion of the repeat and associated tracr-RNA to become g-RNA
- the specific spacer of the crRNA binds to the trcrRNA to form guide RNA (gRNA)
- gRNA then binds with the Cas9 enzyme → forms a Cas9-gRNA complex
- Cas9-gRNA complex scans bacteriophage (target DNA) to look for complementary bases
- once it is found, the DNA is unzipped and Cas9 cuts/cleaves the DNA creating blunt DNA fragments.
- DOUBLE BLUNT END CUT
- the viral DNA cannot reproduce as the DNA has been disrupted
uses of CRISPR Cas9
- scientists can program CRISPR-Cas9 system to TARGET ANY SEQUENCE using the gRNA
- can introduce a sgRNA (single guide RNA) that is complementary to the gene of interest
- knock out genes one at a time, in order to identify their function
- to introduce specific mutations in a DNA sequence
- to edit a faulty allele of a gene in a person with a severe inherited disease
- to snip out the faulty segment of a gene and replace it with a working copy
- to activate or to repress a gene
- to add a new gene to the genome
what is PCR used for
- used to amplify (make more copies) of a segment of DNA
- process depends on the enzyme DNA polymerase → specifically Taq polymerase
- doubles the amount of DNA with EACH CYCLE
steps in PCR
denature
* the DNA sample is heated 94 degrees to 98 degrees for one minute
* this breaks the hydrogen bonds between the double strands
* separates DNA into two single, separate strands
anneal
* PRIMERS are added
* short segments of SINGLE STRANDED DNA
* primers bind to the target DNA (at the complementary sequences), initiating DNA synthesis 50 degrees to 65 degrees for two minutes
extension
* taq polymerase starts at the primers and extends them using free nucleotides
* forms TWO COMPLETE DOUBLE STRANDS
* occurs at around 72 degrees for one minute → optimal temperature of taq polymerase
the process is then repeated
ethical requirements of human experimentation
Respect for persons: Informed consent
* E.g. people must know what they are agreeing to (harms, likelihood of success, etc.)
Integrity: Being honest and trustworthy
* E.g. Being open about the likelihood of benefit or harm
Justice: Selection of human subjects and non-discrimination
* E.g. vulnerable people not targeted
Beneficence: Maximising benefit and minimizing harm
* E.g. the welfare of the person is most important (the research comes second)
Non-maleficence – do no harm
* Eg. Stopping a treatment if it is causing harm
CRISPR Cas9 ethical considerations
- long-term impacts of the technology and informed consent risks
- possibility of the edited genes being inherited and affecting future generations
- the use in individuals who may have not provided consent
- misusing technology such as around the use for designer babies
- confidentiality considerations of genetic data
- personal beliefs and opinions around the editing of human DNA (such as religious or cultural objections).
how can CRISPR Cas9 be programmed to cut any DNA sequence
- scientists create a complex, consisting of a gRNA sequence and the Cas9 protein
- gRNA sequence is complementary to the target DNA sequence
- Using the gRNA, Cas9 identifies the complementary DNA sequence and unwinds the DNA and cuts it
- scientist can insert a new piece of DNA, remove, replace or add or delete single nucleotides at the site of the cut
- the cell detects and repairs the broken strands of DNA
purpose of PAM sequence
- a highly specific region
- a very short nucleotide sequence adjacent to the target spacer (PAM sequence)
- using gRNA, cas9 enzyme searches viral DNA for a PAM
- upon finding it, CAS9 complex compares the sequence of bases in the crRNA to a sequence upstream of the PAM
- If they are complementary, Cas9 cuts the DNA, preventing infection.
- also where Cas9 binds to on the target DNA sequence
- plays an essential role in distinguishing self bacterial DNA from non-self viral DNA
purpose of Cas9 enzyme
- Cas9 will only cut out the target sequence if it recognises a very short nucleotide sequence adjacent to the target spacer called a protospacer
- adjacent motif (PAM) sequence.
- Cas9 is an endonuclease associated with CRISPR and acts likes a pair of molecular scissors capable of precisely cutting both strands of DNA.
GMO definition
genetically modifies organisms are organisms that have had their DNA directly manipulated
transgenic definition
- transgenic organisms are a subset of GMOs that have DNA from different species
- eg. transforming bacteria with recombinant plasmid
how GMO can increase crop productivity
- roundup ready canola
- canola plants are used to make canola oil
- roundup is a herbicide
- roundup ready canola has a bacterial gene inserted
- this allows the canola plant to make an enzyme to break down the herbicide roundup
- GM canola crops can be sprayed with the herbicide and only the weeds die, leaves the GM canola crops unaffected
how GMO can provide pest resistance
- Bt cotton has a bacterial gene that allows it to produce chemicals that will kill insects
- the crops are not eaten by insects → resulting in more cotton production
crops do not need to be sprayed with insecticide
