Molecular biology of gene targeting (6)

Types of gene targeting (2)

1. Forward genetic analysis: disrupt homeostasis by random mutation

2. Reverse genetic analysis: disrupt gene product to assess its function

ES cells

Pluripotent cells/stem cells: that can differentiate into any cell type

Why is HR in ES cells so important?

Bc it allows the creation of knock out mice

1st step

Gene X REPLACEMENT construct

2nd step

HR = neo r mutation

NHR= neo r + tk gene mutations

3rd step

Positive selection of mutated genes with G-418

4th step

Negative selection of tk mutation presenting genes (NHR) with ganciclovir (toxic)

5th step

Injection of HR mutation cells into blastocyst + embryo transfer

Result if process worked properly

Heterogenous progeny bc new phenotype created through the replacement construct

Chimeric embryos

Patched coat color

Hope with HR in pluripotent cells

That the mutation will reach germ cells (oocyte/spermatozoid)

Knockout mouse using the Cre-Lox system

Study of gene by knocking it out only in a specific tissue/specific time

Mice with loxP site

Gene X function normally and has loxP within introns that flank exon 2

Cre mouse

Cre is expressed in different cells and tissues

Crossing of 2 mice=

Knock out specific gene (loxP) in a specific cell (Cre) by excision of exon 2

Transgenic mice

Important for understanding expression patterns and to edit the genomes

CRISPR

Clustered Regularly Interspaced Short Palindromic Regions

What is CRISPR?

Segment of bacteriophage DNA that forms the priRNA

Cas9

Enzyme that:

1. Recognizes tracrRNA on priRNA

2. Recruited to foreign DNA that have crRNA

Activity of Cas9 (2)

1. HNH (3-5)

2. RuvC endonuclease activity (5-3)

TracrRNA+crRNA=

Single guide RNA or sgRNA to a region of the genome

How is the endonuclease cleavage targeted to a specific region?

1. 20 nt homology in general target

2. Homology must be upstream of a Protospacer Adjacent Motif or PAM sequence (NGG)

What happens 3 nt upstream from the NGG segment?

Double strand break

1st step of genome editing

sgRNA from transgene and Cas9 SEPARATE expression = be present in same cell nuclei

2nd step of genome editing

Cas9 recognizes and binds the RNA at target sequence

How is the double strand break repaired? (2)

1. NHEJ

2. HDR

NHEJ

Results in short deletion = induced premature stop codon

HDR

No disruption of the ORF = specific change introduced in the genome