Gene Therapy

How does DNA respond to damage?

1. Checkpoint for further replication

2. Homologous or non homologous end joining repair

3. Self terminate

Homologous repair

A type of double strand break repair. Uses a backup second copy of the DNA (bc we’re diploid) and synthesizes/fixes the DNA strand by using the backup as a template. Does not alter the genes, can be used to edit, add or replace genes!

Non-homologous end joining

A type of double strand break repair. No template sequence used to repair and fill the double stranded break, instead we bring the two end breaks together and repair. Results in deletions in order to bring the strand together, which will insure mutation.

Genome modifications are

Targeted and specific double stranded breaks.

CIRSPR sequences were originally found in bacteria where they

Served as a target for antibiotics because they were the bacteria's adaptive immune system.

Cas-9 protein

Found in the genome of bacteria. after being activated by tracrRNA, ENDONUCLEASE CAS 9 cuts DNA is a double stranded break by finding CrRNA that needs to be cut.

Trans-activating cas-9 RNA

Found upstream of cas-9 protein. Activates cas-9.

How to use CRISPR for genome editing Natural system

1. CRISPR sequence is adaptive immunity sequence, so we artificially insert viral DNA into the sequence.

2. Entire sequence is transcribed into short crRNA

3. Then pair with tracr RNA

4. TracrRNA/crRNA complex then forms a complex with CAS 9

5. Reinfection by artificially adding the viral DNA again

6. CAS9 now will target the virus in its crRNA and cut it once a) tracrRNA activates CAS9 b)cuts

How to use CRISPR for genome editing Engineered guide

1. Instead of crRNA, we have sgRNA which contains the bare minimum of tracrRNA to be able to activate CAS9.

2. Together the complex binds to a target strand that will be upstream of the PAM site.

3. PAM site will be necessary in order for CAS9 to be able to cut about 4 bases upstream of it!

4. Repair the break by end joining OR using backup DNA template for HR

SgRNA

Contains minimum amount of tracrRNA. Guides the CAS9 on where to cut.

PAM site

Downstream of the sgRNA, important for CAS9 to be able to make a cut!

CRISPR for other genetic engineering

Locate dead CAS9 to identify gene location, gene activation, or gene inhibition. Can be attached to proteins with separate functions like a protein that can change base pairing

gel electrophoresis. What does each line represent?

Depending on the prompt given, it may represent mature mRNA or DNA!

Transgenic

Introducing a gene to a different species. First, using reverse transcriptase to get double stranded cDNA. Then, injecting a fertilized egg with the transgenic gene, and causing a germ-line change in the donors gametes, and therefore the altering the genome fate of the offspring. The promoter is solely dependent on the tissue area of expression of the donor, NOT the original promoter.

In vivo

Gene editing or new material delivered made to cells within a person’s body.

In vitro

Modification made to a persons cells after they have been taken out of the persons body.

SiRNA Can be used for

Somatic gene therapy. They are epigenetic, meaning they do not change DNA. They are knockdown type, causing an inhibition of translation of target mRNA.

PCR

Amplify DNA strands in a gel electrophoresis in vitro with cycles of heating and cooling.

Short Tandem Repeats/ VNTRs

Short sequences repeated in DNA in coding AND noncoding regions. Inherited like alleles and vary among all individuals.