BIOL 216 ch3 stem cells and gene editing

iPSCs

adult cells which have been genetically reprogrammed to an embryonic stem cell like state

• express genes and factors which maintain defining properties of ESCs

master regulatory genes

genes expressed in early embryonic stem cells that are induced to be expressed in iPSCs using retroviruses

retrovirus

RNA virus replicated in a host cell via reverse transcriptase to produce DNA from RNA genome

• DNA incorporated into host genome using integrase

• generates cDNA

lentiviral vector

virus that delivers genetic material into cells

• genes that cause disease are deleted

• genes that allow virus to package and deliver genetic material are kept

4 genes added to iPSCs (Yamanaka reprogramming factors)

• Oct4

• Sox2

• Myc

• Klf4

issues with retroviruses

random insertions into the genome can cause mutations and cancer

induction of iPSCs

• somatic cells obtained from adult organism

• Yamanaka factors introduced into cultured cells

• cells grown under embryonic stem cell conditions

• iPSCs grow in colonies

embryonic stem cells

cells can theoretically give rise to all types of cells in an organism

bone marrow stem cells

can generate different types of blood cells and differentiate into bone, cartilage, fat, muscle

stem cell repair

• type I diabetes (insulin from pancreatic cells)

• Parkinson's (dopamine)

• Huntington's (GABA from neurons)

• Alzheimer's (ACh from neurons)

• damaged immune systems (damaged immune cells)

• spinal cord injury (neurons)

• burns/skin grafts (skin cells)

reprogramming a patient's skin cells to be iPSCs

1. remove skin cells from patient

2. reprogram cells to become iPSCs

3. treat iPSCs to differentiate into a specific type

4. return cells to patient

SCNT

• enucleated egg cell receives nucleus from patient cell

• fertilizeed egg develops in a surrogate mother

• remove inner cell mast of blastocyst

• pluripotent stem cells transplant back to patient

CRISPR

segments of prokaryotic DNA containing short repetitions of base sequences interspaced with sequences derived from viruses

• each repetition followed by spacer DNA

Cas9

CRISPR associated 9 nuclease

CRISPR Cas9

immune defense system naturally found in bacteria and archaea

• degradation of invading DNA
• utilized to target locations in complex mammalian genomes and generate site specific breaks in DNA

CRISPR Cas9 uses

• remove a target gene
• insert a donor template into a target region (fix mutation, introduce a tag, create a new restriction site)
• upregulate or downregulate transcriptional activity from promoter region

CRISPR Cas9 process

1. sgRNA designed to complement targeted DNA sequence
2. sgRNA binds to target sequence
3. Cas9 enzyme binds to sgRNA
4. Cas9 makes a double stranded break in DNA
   5a. NHEJ pathway can result in a gene knockout
   5b. HR pathway can create new gene function from a donor gene (knock-in)

sgRNA

RNA acts as a guide for Cas9 enzyme

• combination of crRNA and tracrRNA

crRNA

target specific RNA binds to targeted DNA

tracrRNA

base pairs with crRNA and enables Cas9-crRNA complex to locate targeted DNA

• activates enzymatic function of Cas9

PAM

DNA sequence following target DNA sequence

• necessary for Cas9 to bind and cleave target DNA

NHEJ

repair pathway where DNA ends are ligated back together but usually with the introduction of a small insertion or deletion (gene knockout)

HR

repair pathway where donor DNA with homologous sequences at either end can be integrated into the site (gene knockin)

CRISPR Cas9 influence on cell fate in development

activation of specific endogenous gene expression using CRISPR-mediated activator

• dCas9 (does not cut DNA) and transcription activators can manipulate endogenous gene expression