Manipulating Genomes of Eukaryotes

Forward genetics

begin with a phenotype (mutant individual) and proceed to a gene that encodes a phenotype

Reverse genetics

begin with a gene of unknown function, induce mutation(s) in this gene, observe the effect of mutation(s) on the phenotype of organism

transgenic organism

n organism that has had its DNA altered by adding DNA from another species

Knock-out animal

modify gene sequence to create a loss of function allele

Knock-in animals

add an extra gene into the genome (from the same or different species)

Why make transgenic animals

determine the cellular/molecular function of a gene

model human conditions

analyze localization of specific proteins inside cells

identify a specific gene responsible for the mutant phenotype

rescue experiment

mutation in which gene causes (blank) phenotype?

Analyze gene expression patterns (reporter strains)

GFP is expressed in cells that normally express the gene

cells that normally express the gene are now labeled with GFP and can be visualized

Making transgenic (knock-in) mice by pronuclear injection

site of transgene insertion is random (and usually unknown), injected DNA will randomly integrate into one of the chromosomes

foreign DNA (many copies) injected into one of the pronuclei of a fertilized egg

If integration occurs before first mitosis

all cells of the embryos will carry the transgene

embryos are implanted in a surrogate mother

10-30% of embryos survive; only some will carry a copy of integrated transgene

Targeted mutagenesis

enables alternation of specific genomic sequences

Cas9

CRISPR-Associated protein (endonuclease)- enzyme, makes double stranded breaks in the DNA

sgRNA

single Guide RNA, single-stranded RNA= tacrRNA (binds endonuclease) + crRNA (binds target DNA sequence)

Genome editing by CRISPR/Cas9 step 1

the single guide RNA and Cas9 protein combine to form an effector complex

Genome editing by CRISPR/Cas9 step 2

effector complex associates with the PAM sequence, unwinds the DNA, and pairs with the complementary sequence on the DNA

PAM

protospacer Adjacent Motif (commonly NGG)

Genome editing by CRISPR/Cas9 step 3

the Cas protein cleaves the DNA

The accuracy and efficiency of genome editing are influenced by

double-stranded break repair pathways

non-homologous end joining (NHEJ)

Homology directed repair (HDR)

Non-homologous end joining (NHEJ)

can bring together any DNA ends prone to errors

Homology directed repair (HDR)

accurate repair of double-stranded breaks

requires a DNA template containing the desired edit

Gene therapy

genetic engineering of human somatic cells in order to treat genetic diseases

applying genetic tools to humans

Therapeutic gene

need to know what the disease-causing mutation is and understand the molecular mechanism of a disease (basic science research, model organism)

gene delivery method/vector

efficient delivery of nucleic acids to cells; protection from premature degradation

gene therapy for targeting…

specificity to a tissue/cell type of interest

Viral and nonviral vectors are used

to deliver DNA to specific cells and the nucleus (so it can be expressed!)

delivery to specific tissue in which the transgene is needed is key!

protect the DNA from degradation

Phase 1 clinical trial

focus on safety and the proper dose

Phase 2 clinical trial

focus on effectiveness and side effects

Phase 3 clinical trial

compare the new treatment to existing treatment

FDA reviews the results and can approve the drug for use on people outside the clinical trial

phase 4 clinical trial

treatment approved and available. Long-term effects are observed

look for side effects not seen earlier

study how well a new treatment works over a long period of time

In vivo

therapeutic drug is directly administered to the patient (e.g. injection into target organ)

ex vivo

patient-derived cells are genetically modified in a dish and then re-infused into the patient