Biology 1.6 Biotechnology and CRISPR

Question 1

Biotechnology

Answer 1

The genetic code is universal. This means that genes can be introduced from one species to another, and the gene code will be translated into the same protein.

The manipulation of DNA has powerful and exciting consequences, but ethical concerns must be considered.

Genetic manipulation relies on three basic principles:

1. Gene identification
2. Gene removal
3. Gene ligation

Question 2

GENE IDENTIFICATION

Answer 2

A probe is a short fragment of DNA or RNA that can be radioactively labelled or fluorescently labelled.

Labelled probes can then be used to identify specific complementary nucleotide sequences in DNA or RNA

Question 3

GENE REMOVAL

Answer 3

Restriction enzymes recognise specific sequences in DNA and cut through the sugar-phosphate backbone.

The target sequence is called the recognition site.

The cut in the DNA may leave overhanging ‘sticky ends’.

Question 4

GENE LIGATION

Answer 4

Fragments of DNA with complementary sticky ends can be joined/ligated together using the enzyme ligase.

Question 5

transferal of genes BETWEEN species

Answer 5

Genes can be transferred between species using techniques including:

1. Vectors

Plasmids

Viruses

2. Electroporation
3. Microinjection

Question 6

VECTORS: Plasmids

Answer 6

The gene of interest is identified in another organism using a labelled probe.

The plasmid and gene of interest are cut with the same restriction enzymes.

This produces complementary ‘sticky ends’ in both the plasmid and gene of interest. This allows them to be joined together.

Ligase joins the DNA sections, and the genetically modified plasmid is returned to the bacterial cell.

Question 7

VECTORS: Viruses

Answer 7

Viruses are surrounded by a protein layer (capsid) and contain a small amount of DNA.

Viruses can be engineered so that they can enter cells and release their DNA, but not replicate to produce more viruses.

They can also be used to deliver foreign DNA into human cells for gene therapy. The foreign, functional gene can then be expressed as protein.

Question 8

ELECTROPORATION

Answer 8

In electroporation, an electrical pulse is used to create small holes in cell membranes.

This allows DNA and/or RNA to be delivered to the target cells.

This method is versatile, and can be used in yeast, bacteria, and ‘stripped’ plant cells.

Question 9

MICROINJECTION

Answer 9

In DNA microinjection, a very fine glass pipette is used to manually inject DNA from one organism into the eggs of another.

Time of injection is important and is best early after fertilisation.

DNA is not always successfully incorporated into the genome. As a result of this, it is possible the gene insert will not be expressed by the genetically modified organism.

Question 10

transferal of gene WITHIN species

Answer 10

Genes can be edited and/or transferred within a species using a technique called CRISPR.

Question 11

CRISPR

Answer 11

Bacteria use CRISPR to fight off viruses (bacteriophages) by cutting up viral DNA and storing it, so they can ‘remember’ how to fight future invaders.

Scientists have adapted this system for use in gene editing and transferal in a range of organisms.

CRISPR is a powerful technique that has a wide range of applications, including the treatment of disease.

There are two key components of the CRISPR-Cas9 system:

Cas9
An enzyme that cuts DNA (endonuclease).

Guide RNA
Complementary to a target sequence.
Directs Cas9 to the target to cut the DNA.

Question 12

CRISPR

Answer 12

Inside a cell, Cas9 and the guide RNA (gRNA) form a complex.

Cas9/gRNA scan the DNA inside the cell, looking for a protospacer adjacent motif (PAM). The complex unzips the DNA and allows the gRNA to bind to the DNA via complementary base pairing.

Cas9 then makes two cuts in the DNA (one in each DNA strand).

The cell tried to repair the break, but makes errors, inactivating the gene.

OR

A new sequence can be introduced.

Question 13

CRISPR Advantages

Answer 13

Cas9 has many variants that can be used in a variety of studies (versatile).

Very simple and efficient technique.

Can be applied directly in an embryo, therefore reduces time required to modify target genes.

Experimental conditions can be optimised (eg: identifying the most appropriate gRNA sequences) to aid in successful introduction of desired mutation.

Question 14

CRISPR Limitations

Answer 14

Potential lack of target specificity and accuracy (leading to unforeseen complications). Scientists currently looking for alternatives to the Cas9 endonuclease.

Difficult to deliver CRISPR/Cas material to cells in large numbers (not efficient).

Ethical concerns regarding genome editing – potentially leads to “designer babies”.*