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GENOME EDITING

Genetic approaches

• How do we identify a gene as being involved in a disease

or process?

• Forward genetics -> mutagenise a population of

individuals or cells, screen for phenotype of interest.

• Reverse genetics -> Gene identified as potentially involved

in disease, process -> modify to analyse function.

• GWAS studies -> Reverse genetics.

Genetic engineering

• Recombination approaches (mice, yeast).

• Transposable elements ( Drosophila ), Bacterial insertion

of DNA ( Arabidopsis ).

• Transfection of gene constructs into cells (randomly

integrated).

• Until recently, not possible to make genetic changes in

human cells -> genome editing.

What is genome editing?

• (^) A genetic engineering approach in which DNA is inserted,

removed or replaced at a precise location within the genome.

• (^) Engineered nucleases.
• (^) Recombination-based approaches

Nuclease based genome editing:

• Zinc-finger nucleases
• Meganucleases
• TALENs
• CRISPRs

Viral genome editing:

rAAV

(Recombinant adeno-associated

virus)

This occurs without dsDNA break

by homologous recombination

• (^) Creation of isogenic cell lines -> only differ by the change

we’ve introduced.

• (^) Make (nearly) any modification we desire!

Genome editing

Precise alterations to the genome Single base pair changes (^) Knock-out rAAV CRISPR (^) TALENs Insertion A gene knockout is when a gene has acquired a frameshift mutation such that it no longer expresses any functional protein

Nuclease-based genome editing

TALENs - transcription activator-like effector nucleases

Xanthomonas bacteria express TAL arrays to bind and activate host promoters TAL array is a series of DNA binding domains assembled to recognise a specific sequence 33-34 amino acid sequence – only 12th^ and 13th^ residue vary – and determine nucleotide binding. We can construct these arrays, add a nuclease and use for genome editing Engineered nucleases which will cut at a desired position in the genome TGAGGAGGCGGCAACG GCGGGCGCCGGGGCGG CGGGCCCCGGGGCGAGCA ACTCCTCCGCCGTTGC CGCCCGCGGCCCCGCC GCCCGGGGCCCCGCTCGT Fok Fok Cleavage

CRISPRs (Clustered Regularly Interspaced Short Palindromic

Repeats)

CRISPR-Cas system – an form of acquired immunity found in bacteria. The guide RNA directs the Cas9 protein to a target site. Creating a guide RNA is very simple. Cas9 Protein Cleavage Guide RNA

Nuclease-based genome editing

CRISPR/Cas9.

CRISPR/Cas9 is a system found in bacteria and involved in immune defence. Bacteria use CRISPR/Cas9 to cut up the DNA of invading bacterial viruses that might otherwise kill them.

Emmanuelle Charpentier and Jennifer Doudna share the Nobel Chemistry prize in 2020 for developing the precise genome-editing technology.

Nuclease-based genome editing

TALENs and CRISPRS Nuclease-induced DSB NHEJ-mediated repair (non-homologous end joining) Insertion or deletion mutations HDR-mediated repair (Homology directed repair) Single nucleotide alterations Donor Template Donor Template Precise sequence insertion