CRISPR

Question 1

Discovery of CRISPR

Answer 1

Noticed that some bacteria had DNA changes in highly repetitive sequences with neighboring CRISPR-associated (Cas) genes in their vicinity
Discovered that CRISPR-spacers matched phage DNA: incorporation of viral DNA into bacterial genome in order to use viral sequence to attack virus

Question 2

Components of CRISPR system: protospacer adjacent motif (PAM)

Answer 2

Part of target sequence that Cas9 binds to (not gene of interest)
Has 2 G’s in a row

Question 3

Components of CRISPR system: Cas9

Answer 3

Nuclease that induces double strand break of DNA to be edited

Question 4

Components of CRISPR system: scaffold sequence

Answer 4

3’ end of target sequence
Part of guide RNA
Bound by Cas9

Question 5

Components of CRISPR system: target sequence

Answer 5

Part of guide RNA

Complementary to gene of interest

Question 6

Components of CRISPR system: donor template

Answer 6

Sequence that contains gene to be inserted

Following double strand break by Cas9, donor template is used to repair cut, resulting in gene insertion

Question 7

2 outcomes of Cas9-mediated double strand break

Answer 7

Non-homologous end joining (results in indels, which are insertions or deletions that typically cause knock-outs)
Homology-directed repair: template is used to repair cut, resulting in precise edits (gene insertion)

Question 8

Cpf1

Answer 8

Similar to Cas9, but uses NTT PAM
Smaller than Cas9 with guide RNA that is half as long
Avoids triggering immune response (sometimes happens with Cas9)

Question 9

dCas9

Answer 9

Cas9 without nuclease activity
Can repress genes by being in the way of RNA polymerase
Can be fused to effector domains for gene activation, chromatin modification, GFP, etc.
Can be combined with DNA modifying enzymes to directly edit bases without repair template
Can alter just one of two nuclease activities so that single cut is made for higher base editing efficiency

Question 10

Massively parallel genome wide screen

Answer 10

Use of CRISPR system to analyze effects of many genes

Ex- knock out every gene in genome or specific region

Question 11

CRISPR for gene therapy

Answer 11

Isolate cells from patient -> make desired edit to repair disase-causing mutation -> reinsert repaired cells into body

Question 12

CRISPR and Duchenne muscular dystrophy

Answer 12

Use gRNA to execute exon skipping: correct frame shifts or premature stops in dystrophin gene
Corrected induced pluripotent stem cells are turned into heart muscle cells

Question 13

CRISPR and embryos

Answer 13

Corrected beta-thalassemia in embryos: gRNA sequences targeted hemoglobin B gene

Question 14

Problems with Cas9-mediated gene editing of HBB (hemoglobin B gene)

Answer 14

Efficiency of homologous recombination directed repair of HBB was low and edited embryos were mosaic (cells can repair using other pre-existing sequences rather than donor template)
Off-target cleavage was present
Endogenous delta-globin gene (homologous to hemoglobin B gene) competed with exogenous donor oligos to act as repair template, leading to unwanted mutations from recombination products

Question 15

Reasons for human germline editing

Answer 15

Correction of disease
Enhancement of human traits (intelligence, attractiveness, etc.)
Protection from disease (CRISPR babies: HIV resistant)

Question 16

Reasons against human germline editing

Answer 16

Technical issues (not totally efficient, human cells tend to prefer non-homologous end joining over homology directed repair)
Ethical issues