Gene Editing

Question 1

genome-editing

Answer 1

use CRISPR/Cas9 as “DNA scissors” to cut genome
remove, change, and add DNA

Question 2

genomics

Answer 2

study of entirety of an organism’s genes - genome
use bioinformatics to analyze DNA-sequence data to find variations

Question 3

genome-wide association study

Answer 3

in genetics research - associate specific genetic variations with particular diseases

Question 4

gene editing technologies

Answer 4

ZFN - directed evolution
TALEN - clone new RVDs
→ a string of DNA recognition protein motifs is designed and fused with cleaving enzyme

CRISPR/Cas9
→ generate gRNA that binds to target sequence

Question 5

functional genomics

Answer 5

gene editing to address a disease mutation

Question 6

CRISPR

Answer 6

clustered regularly interspaced palindromic repeats

found first in E.coli, repeat sequences and spacers
bacterial defence system against viral infections

Question 7

CRISPR/Cas9 pathway

Answer 7

prokaryotic immune system that defends against viruses
1. virus infects bacteria
2. incorporated into CRISPR locus
3. trancription of CRISPR into pre-crRNA
4. form complex with guide RNA
5. Cas9 - scissors that cut DNA, guided by gRNA
6. another infection - targeted with CRISPR/Cas9 system
7. Cas9 protein cleaves invading DNA

Question 8

Cas9 recruitment

Answer 8

1. Cas9 DNA endonuclease
2. crRNA - contains a 20 bp sequence complementary to target
3. trans-activating crRNA (tracrRNA) - acts as a bridge between crRNA and Cas9 enzyme

Question 9

mechanism of DNA cleavage with CRISPR/Cas9

Answer 9

sgRNA → DNA endonuclease is targeted to a DNA sequence upstream of PAM = double strand break
DSBs are repaired by NHEJ or HDR

insertions or replacements can be incorporated into break

Question 10

off-target DNA cleavage

Answer 10

cutting = DSB → mutations
guide sequence can target even 1-2 nucleotide difference

1-bp insertion = DNA bulge
1-bp deletion = RNA bulge

Question 11

design crRNA spacer sequence

Answer 11

1. identify PAM sequence in DNA to target
2. determine 5’ start of sgRNA targeting sequence → 20 nt upstream of PAM
3. determine sgRNA sequence

3’ end of DNA target sequence must have PAM sequence
20 nt = target sequence → Cas9 will cleave ~3 bases upstream of PAM

Question 12

PAM

Answer 12

proto-spacer adjacent motif sequence
NGG → any nucleotide + two guanines (ex. TGG)
required for cleavage but is not part of sgRNA sequence

Question 13

delivering CRISPR/Cas9

Answer 13

Cas9 can be delivered as DNA or mRNA molecule encoding Cas9 gene; or functional ribonucloprotein

challenge: delivering it across cell membrane

Question 14

viral vectors

Answer 14

AAV - carries DNA
useful but challenge in immune response

Question 15

non-viral vectors

Answer 15

physical methods: microinjection and electroporation (DNA, mRNA, protein/RNP)

chemical methods: Cas9-CPP/NLS, LNP, AuNP → safer

Question 16

gene editing applications

Answer 16

double stranded breaks created with CRISPR/Cas9
HDR uses ssDNA donor template → insert new DNA
= precise gene knock-in

Question 17

base editing

Answer 17

1. Cas nickase/Cas fused to deaminase - make edit
2. gRNA targeting Cas to specific locus
3. target base for editing

base excision repair inhibitor is also present in fusion protein (solves off-target effects)
only makes small nick → changes single base pair

deamination of C to U or A to I (G) → transition mutations after DNA replication

Question 18

prime editing

Answer 18

engineered reverse transcriptase fused to Cas9 nickase + prime-editing guide RNA
from RNA to DNA
make small insertions/deletions in DNA

Question 19

gene activation

Answer 19

use CRISPRa → can target promoter to enhance gene expression
dCas9 (inactive) can be combined with other activators of genes (fusion to transcription activator)

Question 20

gene repression

Answer 20

use CRISPRi → interference to inhibit gene expression
dCas9 (inactive) is fused to a transcription repressor

Question 21

epigenome editing

Answer 21

dCas9 fused to core catalytic domain of p300 → can acetylate target sites in the genome
acetylation of target gene synergizes with action of transcription factors and RNA pol II = transcriptional upregulation

p300 releases the coils of histones in genes to increase expression

Question 22

CRISPR screening

Answer 22

capable of making highly specific, permanent genetic modifications → more likely to ablate target gene function
used extensively to screen for novel genes that regulate known phenotypes (ex. resistance to chemo drugs, toxins; cell viability; tumor metastasis)

gRNA → synthesized gRNA oligos → CRISPR library → amplified → use vector to transfer to lentivirus + Cas9 expressing cells → infect other cells with library → positive/negative selection

Question 23

example: dystrophin protein

Answer 23

single cut CRISPR strategy restores dystrophin protein
mutation in exon 50 = 49 and 51 don’t fit together
Cas9 → in frame-deletion = restores frame and function

Question 24

ex vivo therapy

Answer 24

in vitro
isolate blood cells → correction with CRISPR → clonal selection + expansion → return to body

useful for blood diseases - sickle cell anemia, hemophilia, CAR T cancer

Question 25

in vivo therapy

Answer 25

CRISPR/Cas9 + HR template → viral or non-viral delivery → systemically treat

useful for diseases of eye and ear - clinical trials

Question 26

somatic gene editing

Answer 26

edit - target genes in specific types of cells (ex. blood cells) → any cell other than reproductive
copy - edited gene is contained only in target cell type

any changes are limited to treated individual = edited gene is not heritable

Question 27

germline gene editing

Answer 27

edit - germline modifications are made early in development → changes are copied into all new cells including sperm or eggs

edited gene is heritable → pass to future generations
consideration of ethical and legal issues