BIO 2.2-2.7 - Enzymes, CRISPR-Cas9, PCR, gel electrophoresis and GMOs

CRISPR

Clustered regularly interspaced short palindromic repeat

Cas9

An endonuclease that acts as molecular scissors that can cut two strands of DNA at a specific location to remove or add DNA

Single guide RNA (sgRNA)

A piece of RNA consisting of CRISPR RNA (crRNA) that is a 20 base long sequence that is complementary to the target DNA and trans-activating CRISPR RNA (tracrRNA) which is a binding scaffold for Cas9

PAM sequence

The protospacer adjacent motif is a short DNA sequence that is downstream from the target DNA sequence (towards the 3’ end). The PAM is the binding site for Cas9, which allows the enzyme to cut the correct part of the DNA, distinguishing self from non-self

Steps of how CRISPR-Cas9 works

1. Scientists create a crRNA sequence that is complementary to the target DNA sequence

2. Cas 9 forms a complex with sgRNA

3. This complex identifies the correct PAM sequence and binds to the DNA

4. The Cas9 unwinds the DNA and the sgRNA complementary base pairs with the target DNA sequence

5. The Cas9 enzyme cuts both of the DNA strands upstream of the PAM sequence, leaving blunt ends

6. The DNA is edited by inserting, deleting or disabling nucleotides

7. Once the DNA is repaired, it is incorporated into the genomic DNA of the organism

Steps of how CRISPR-Cas9 works in bacteria to defend against viruses

1. A virus attaches and injects viral DNA into the bacterial cell

2. Cas2 endonuclease cuts up the viral DNA and inserts a section of the viral DNA (spacer) into the bacterial CRISPR set of genes beside a repeat section

3. When the virus attacks again, the viral DNA spacer transcribes the gRNA matching the viral DNA and Cas9 is produced, forming a complex

4. The Cas9 complex attaches to the viral DNA and chops it up

Enzymes

Proteins that catalyse chemical reactions to build or break molecules

DNA polymerase

Synthesises new DNA using each strand of the DNA double helix as a template

Taq polymerase

A special type of DNA polymerase extracted from a thermophilic bacterium. It has a high optimum temperature which is utilised in processes such as PCR.

Reverse transcriptase

Synthesises complementary DNA (cDNA) from an mRNA strand, reversing the transcription process. It is used to insert a gene without introns into a plasmid.

Restriction endonucleases

DNA cutting enzymes that cut phospodiester bonds between nucleotides. The enzymes recognise a specific sequence of nucleotides, bind to the recognition site and cut the DNA at that site to make sticky ends or blunt ends.

DNA ligase

Catalyses the formation of phosphodiester bonds to join nucleotides together

PCR

Stands for polymerase chain reaction. PCR is a process in which DNA polymerase is used to copy a DNA sequence repeatedly, making millions of copies of the same DNA (amplification)

Substances placed inside the PCR test tube

• Sample of DNA to be copied

• The 4 nucleotides (ATCG)

• Taq polymerase

• Two types of single stranded DNA primers which are synthetic, short pieces of DNA that are complementary to the sequence of bases that flank the target DNA region

Steps of PCR (different temperatures)

1. Denaturation: The mixture is heated for 30 seconds to 95°C to separate the complementary nucleotides to produce 2 single stranded templates

2. Annealing: The temperature is lowered to 55°C which allows the primers to bind (anneal) to the DNA templates

3. Extension: The temperature is raised to 72°C, which is the optimal temperature for taq polymerase, which moves along the template, adding nucleotides complementary to the target sequence in a 5’ to 3’ direction

4. 2 double stranded DNA molecules are produced and another round of replication begins

Gel electrophoresis

A process that separates fragments of DNA based on their size and charge

Steps of gel electrophoresis (6)

1. Restriction enzymes cut DNA into small fragments called STRs (short tandem repeats)

2. The STRs are amplified using PCR

3. A buffer solution is poured over the agarose gel in the chamber

4. STRS are loaded into the wells at the negative end of the agarose gel

5. Electric current is passed through the chamber, causing the negatively charged DNA to move towards the positive terminal. Smaller fragments move faster and further than larger fragments, sorting DNA fragments by size

6. The gel is placed under a UV light to view the fragments appearing as bands

STRs

Short tandem repeats are sequences of 2-6 bases that are repeated, being inherited from parents

Polymorphism

Refers to how the length of short tandem repeats varies between individuals, allowing for the identification of an individual by profiling their STRs using gel electrophoresis. i.e. no two individuals will have the exact same number of repeats for 13 STRs.

Applications of gel electrophoresis

DNA profiling to determine paternity, suspects of a crime and risk of genetic disease

Genetically modified organism

An organism that has had its genome altered using genetic engineering technology.

A genome can be altered by adding genes or silencing genes.

Transgenic organism

An organism that has had genes from another species inserted into its genome (e.g. inserting a gene from an apple into an orange)

Steps of inserting genes into plant cells

Plant cells have a cell wall, meaning a bacteria called Argobacterium tumefaciens must be used to infect plant cells

1. Recombinant plasmids carrying the desired gene are transformed into the bacteria

2. The transformed bacterial cells are cultured with plant cells

3. The plant cells become infected by the bacteria and now contain the plasmid

4. The infected plant cells are cultured to make new plants with the new gene to grow a genetically modified crop

Examples of genetic modification

• Herbicide resistance

• Pest resistance

• Virus resistance

• Drought, flood and salt tolerance

• Enhanced nutritional value (golden rice)

Biological implications of genetic modification

• Gene transfers could be harmful to an organism

• Releasing GMOs into the environment could be uncontrollable

• Cross pollination between GM and non-GM crops

• Reduces genetic variation

Social implications of genetic modification

• Solves malnutrition and hunger

• Creates more social inequality due to the high cost of the technologies

• Consumer choice of consuming GM foods

• Changes to business practices and the economy

Ethical implications of genetic modification

• Patents for the ownership of the genome of GM crops

• Violation of animal rights

• Introducing human genes into animals - does the animal gain human rights?

• Intervention in the evolutionary process