W - gene therapy and genetic ethics

DNA repair for double stranded breaks

nonhomologous end joining and homology directed repair

NHEJ repairs double stranded breaks, but

often introduces mutations

NHEJ can

join two ends directly together, remove a few nucleotides first, or add in random extra nucleotides first

Inducing a DSB at a ____ ____ can lead to random mutations if NHEJ is used

precise location

Random indels can be introduced at the targeted site

to generate LOF mutations, which relies on cutting the gene in a place where a random indel is likely to disrupt function

targeted mutagenesis 

generates random point mutations is a specific region

The DSB will be repaired based on a _____ (same sequence) repair template.

homologous

HDR normally relies on a ____ ____. BUT…if a repair template with _____ ___ is provided, the cell may use that instead

sister chromatids, homology arms

HDR uses a homologous template to “___ in the ___” of what was lost

fill, blank

The homology template determines what the sequence between homology arms will be ____ repair.

after

CRISPR-Cas9

bacterial defense system against viruses

Bacteria integrate bits of ___ genetic material into their genomes.

viral

Viral integrated parts encode for RNAs that can bind to viral DNA by complementary base pairing, allowing the bacteria to ____ and _____ viruses.

detect, destroy

The ____ ___ directs Cas9 to cut at

specific locations based on base

pairing between the ____ ____

and the ___ _____

guide RNA, spacer region, target sequence

Cas9

enzyme that cuts DNA to form double-stranded breaks

Scientists adapted CRISPR-Cas9 for biotechnology by creating a _______

single guide RNA

CRISPR-Cas9 is ____ AND ______,

which enables _____ genome editing

specific, programmable, targeted

specific

it will cut only at particular a DNA sequence

programmable

The DNA sequence it cuts is not set in stone, but rather can be determined (by the sequence of the guide)

restriction enzymes are NOT 

programmable 

1st condition for Cas9 to cut DNA

The single guide RNA base pairs to the DNA

2nd condition for Cas9 to cut DNA

The DNA in the non-complementary strand contains a PAM

sequence in the correct location

correct location of PAM sequence

directly adjacent to where the guide binds, on the side closest to the 3’ end of the guide

If a guide contains the PAM, that part of the guide also

base pairs to the ___ strand

top

guide RNA

lies directly upstream of the PAM sequence with Us instead of Ts

For targeted mutagenesis, you just need

Single guide RNA, Cas9

For targeted editing, you just need

Single guide RNA, Cas9, homologous repair template

Hemoglobin

protein in red blood cells that carries oxygen

hemoglobin is a tetramer of

2 alpha-globin and 2 beta globin (each subunit is encoded by its own gene)

missense mutation in beta-globin gene

changes the 6th amino acid from glutamate to valine, which causes protein aggregation and sickle cell disease

null/amorphic mutation in beta-globin gene

reduces the amount of β-globin produced causes to β-thalassemia

fetal hemoglobin

incorporates gamma-globing instead of beta-globin

Around birth, γ-globin expression mostly

turns off and β-globin begins being expressed

All blood cells are derived from blood stem cells in the

bone marrow

Bone marrow transplants

A recipient’s stem cells are purposefully killed by chemotherapy, then replaced by donor cells.t

transplant process

collection, processing/purification/concentration, cryopreservation (freezing), chemotherapy, reinfusion

Allogenic

(from a donor) bone marrow transplants depend on matching to a suitable donor

Autologous

(from self) bone marrow transplants do not risk rejection

BCL11A

TF that promotes the switch from expressing γ-globin (fetal) to β-globin (adult)

In fetuses, a ____________ interaction promotes γ-globin

expression

TF/coactivator/enhancer

Binding to the γ-globin promoter ____ the activating complex from forming. The complex forms on the ______ promoter instead.

prevents, β-globin

Casgevy introduces mutations in an enhancer of

BCL11A (This enhancer regulates BCL11A gene expression, it is not one of the enhancers that BCL11A protein binds to)

1st advantage of Autologous CRISPR therapy

Targeted mutagenesis is easier than gene knock-in (doesn’t require homology-directed repair with a template—just break an enhancer!)

2nd advantage of Autologous CRISPR therapy

Casgevy works for multiple kinds of β-globin disorders regardless of what is wrong with a particular patient’s β-globin gene

3rd

Using a patient’s own [edited] marrow cells makes transplant rejection unlikely

Somatic genome editing has been used to develop human gene therapies, but _____ editing is widely banned

germline

positive eugenics

seeks to increase fitness by the deliberate enrichment of “good genes”

negative eugenics

seeks to increase fitness by stopping the spread the of “bad genes”