molecular biology of gene targeting

identification of gene function

• function is best addressed through removal of gene activity and analysis of the resulting phenotype

• abnormal phenotypes indicate specific processes have been disrupted that rely on the activity of the affected gene

removal of gene activity

• disrupt homeostasis based on random mutation (forward genetic analysis)

• disrupt the activity of specific gene product to assess its function (reverse genetic analysis)

homologous recombination of pluripotent stem cells

• gene replacement construct (containing positive and negative selection markers) is introduced into embryonic stem cells

• if homologous recombination occurs, neo^r (positive selection marker) is inserted into the target gene X and cells become resistant to G-418

• if nonhomologous recombination occurs, neo^r and tk^HSV (negative selection marker) are randomly inserted into the genome and cells become resistant to G-418 but sensitive to ganciclovir

• only cells that have undergone homologous recombination can survive the two selection steps

ganciclovir

toxic in the presence of the herpes virus tk gene, so it will negatively select against all non-homologous recombination events in pluripotent stem cells

embryonic stem cells following homologous recombination with replacement construct

• ES cells are used to populate and blastocyst of an acceptor mouse, this mouse strain has to be another coat colour that is recessive

• the blastocyst is transferred to a surrogate mouse mother

• progeny will be a mixture of both genotypes if the cells were viable and the process worked properly

• population of totipotent cells is therefore heterogenous in the blastocyst

• homogenous hist embryos will give rise to black mice

• heterogenous (host + targeted cells) will be chimeric (spotted/patched coat colour)

• implanted cells contribute to various tissues

Cre-Lox system of cell type-specific knockout

• useful to study the function of a gene in a specific developmental event

• enables study of the function of a gene that is essential for viability by knocking it out only in a specific tissue or at a specific time (Cre-ER(LBD))

loxP mouse in Cre-Lox system

has loxP sites introduce within introns flanking an exon of the target gene

Cre mouse in Cre-Lox system

• express Cre in cells determined by the adjacent promoter

• different strains of Cre mice express Cre in different types of cells and tissues

loxP-Cre mouse in Cre-Lox system

target gene is knocked out in the specific cell type of interest due to interactions between loxP sites and Cre protein

benefits of transgenic mice

• transgenes integrate randomly into the genome so positional effects may affect gene expression

• transgenic reporter genes are important for understanding expression pattern of genes

• can express transgenes under endogenous promoter or heterogenous control

• can be used to edit the genome

CRISPR/Cas9 as a bacterial acquired immune response

• segments of bacteriophage DNA sequence are integrated into the genome of some species of bacteria in clustered regularly interspaced short palindromic regions

• regions are transcribed into primary RNA that is bound by tracr/trRNA

• Cas9 recognizes structures in the tracrRNA and is recruited to foreign DNA segments that are recognized by the crRNA

• Cas9 possesses both HNH and RuvC-like endonuclease activity

CRISPR/Cas9 for genomic engineering/editing

• combination of crRNA and tracrRNA can be expressed as a single guide RNA (sgRNA) that recruits Cas9 to a region of the genome (separate transgenes needed for sgRNA and Cas9)

• targeting is achieved through 20nt homology to a DNA gene target upstream of a protospacer adjacent motif (PAM) sequence (NGG)

• Cas9 will generate a double-stranded break (using two separate endonuclease domain) 3nt upstream of the PAM sequence in the target DNA

resolution of double-stranded break by CRISPR/Cas9 with nonhomologous end joining

• short deletion disrupts open reading frame (ORF)

• error-prone, often introducing frameshifts, giving rise to mutations

resolution of double-stranded break by CRISPR/Cas9 with homology-directed repair

• repair template transgene is intentionally introduced

• gene targeted by CRISPR/Cas9 is replaced, specific change introduced to the genomic DNA