lecture 23: Gene therapy + CRISPR

bio 302 exam 4 material

monogenic

human diseases that are due to a defect in a single gene

• if a good copy of that gene could be introduced into the genome of cells lacking it, it may “cure” the disease

somatic cell therapy

introducing a human gene into somatic cells to treat a patient with a disease

retroviral vectors

the therapeutic gene is inserted into a disarmed retrovirus (RNA virus); engineered virus is used to infect cells from patient, its RNA genome is copied into dsDNA by reverse transcriptase and integrates into host cell genome

pro

pro/con of using other viral vectors

lack of viral integration reduces risk of insertional mutagenesis

con

pro/con of using other viral vectors

lack of viral integration means constant injections of cells is required

spinal muscular atrophy 1

an autosomal recessive disease in the SMN1 gene when untreated is lethal by age 2

germline gene therapy

introducing a human gene into the germline of a patient with a genetic disease to “cure” the disease in the offspring

CRISPR

allows the precise editing of genes in their normal place in the genome in living cells or organisms w/o permanently introducing any foreign genes

• resulting genes are not transgenic

• normal role: bacterial viral defense system

• can be used to mutate or change any gene if you know the sequence of and whuch

Cas protein

cuts the invading DNA next to the PAM (short 3bp site) and becomes the new spacer in the DNA array

• if it sees that same phage again, the Cas protein uses an RNA version of the spacer to recognize the phage DNA genome and cut it up

spacer sequences

used as a memory to protect the bacteria from any phage that it or its ancestors have been infected by in the past

Cas 9 system

find a 3 bp PAM site near the region you wish to cut; make a small RNA with a complementary sequence (guide RNA)

• inject guide RNA along with Cas9 protein into your organism and that DNA site in the genome is cut by the Cas protein

nonhomologous end joining (NHEJ)

after cleavage of dsDNA by CRISPR, ends are joined back together, but this can sometimes introduce deletions (make mutations)

homology-directed repair

after cleavage of dsDNA by CRISPR, the cell repairs a break in a chromosome by using the sequence from other homolog to fill in the gap

• if we put a DNA fragment inside the cell, it will use that instead

• we can make the changes we want

d) all three

what do you need in order to perform CRISPR?

a) cas protein and specific guide RNA

b) case protein

c) donor dna w/ change you want + specific guide RNA

d) donor dna + specific guide RNA + cas protein

preimplantation genetic diagnosis

carriers of genetic disease can reproduce by IVF and the resulting embryos can be tested to see if they carry or lack the disease genes

• can also be done to select ‘desirable properties’