lec 7 - CRISPR beyond gene editing

Question 1

editing out disease

Answer 1

• sgRNA into eukaryote –> nuclear localization signal gets it into nucleus –> guide recruites cas9 to site –> induces dsDNA break –> DNA machinery repairs break (NHEJ or HDR)

Question 2

editing out disease

sick cell disease

Answer 2

• sickle cell due to missense mutation
• HDR targets and repairs faulty gene
• cas9 promotes fetal hemoglobin by breaking down repressor gene (BCL11A) –> NHEJ (common)

Question 3

duchenne muscular dystrophy

Answer 3

NHEJ for exon skipping

Question 4

backer muscular dystrophy

Answer 4

• huge deletions but milder symptoms because in-frame deletions –> shorter yet functional
• skip exons to reframe to original reading frame thru NHEJ

Question 5

antiviral applications

HIV

Answer 5

• challenge because it has continuous reservoir in host
• edit host: prevent HIV entry into CD4+ T cells
• target virus: exon skipping to pluck disease out

Question 6

code cracking

Answer 6

functionally annotate non-coding regions –> identify microRNAs –> find function

Question 7

antimicrobial applications

program bacterial death

Answer 7

engineer self-targeting CRISPR systems –> sequence-specific antibiotics

Question 8

program bacterial death

advantages (3)

Answer 8

• precisely target genotypes
• sgRNAs enable wide range of organisms to be targeted at same time
• precise eradication of pathogens –> survival of beneficial commensal bacteria

Question 9

program bacterial death

challenge

Answer 9

must develop robust delivery options

Question 10

antimicrobial applications

phage therapy

Answer 10

introduce cas9 into bacteriophage –> sgRNA targets antibiotics resistance gene –> bacteria becomes sensitive to antibacteria –> dies

Question 11

industrial applications

in bacteria (3)

Answer 11

• genotyping
• vaccinating industrial cultures against viruses
• controlling uptake and dissemination of antibiotic resistance genes

Question 12

industrial applications

for agriculture

Answer 12

manufacture green chemicals (biofuels and biomaterials)

Question 13

basic research applications

creating transgenic animals (traditional way)

Answer 13

harvest inner mast cells from embryo in blastocyst stage –> induce homologous end joining –> inject back into animal

Question 14

basic research applications

creating transgenic animals (with CRISPR)

Answer 14

directly introduce cas9 into embryo –> germline transmission (faster)

Question 15

multiplexing

Answer 15

knock down multiple genes at once

Question 16

basic research applications

what challenges did it look like they overcame?

Answer 16

• off-target mutations
• mosaics
• dsDNA breaks

Question 17

basic research applications

what problems were still hidden underneath?

Answer 17

• dsDNA breaks looked heterozygous but were homozygous –> genotoxicity
• HDR is bad in early human embryos –> avoid CRISPR in embryo

Question 18

list (5)

CRISPR challenges

Answer 18

• accuracy and efficiency of cleavage and repair
• minimize/eliminate off target effects
• efficient delivery to specific cell types, tissues, organs
• understand how to control various repair pathways
• predict mutational outcomes of DNA repair after DSB

Question 19

list (5)

expand genetic toolbox

Answer 19

• extend PAM dependent targeting space –> reach every base in genome
• reduce off-target binding
• increase specificity
• increase efficiency
• reduce size

Question 20

expand toolbox

xcas9

Answer 20

• proein engineer xCas9 variant
• uses PAM site NGN where N can be any base, instead of NGG
• reaches more bases

Question 21

expand toolbox

mini-CRISPR

Answer 21

search for what’s out in nature

Question 22

expand toolbox

small chemicals

Answer 22

• inhibit/promote NHEJ/HDR or or knockdown genes that do that

Question 23

deas cas9

Answer 23

• mutated cas9 that lacks DNA cleavage activity but retains DNA binding activity
• fuse it to transcriptional repressors or activators of target genes

Question 24

CRISPRi

Answer 24

inhibits transcription of target genes

Question 25

CRISPRa

Answer 25

activates transcription of target genes

Question 26

genome wide screens; CRISPRi/a functions

Answer 26

- knocks down every gene in genome --> determines genes important to us --> design sgRNA to target every gene - CRISPRi: silences genes by targeting promoter region - CRISPRa: gain of function mutations --> amplify genes to determine function

Question 27

epigenetics

Answer 27

- modify epigenetic marks to determine their function - long lasting effects, no genotoxicity (dsDNA breaks)

Question 28

base editors

Answer 28

- change nucleotide bases --> pinpoint repairs

Question 29

# base editor dead cas9 + adenosine deaminase

Answer 29

- converts adenosine to inosin (guanine) --> A-T to G-C - corrects missense mutation

Question 30

# base editor dead cas9 + cytosine deaminase

Answer 30

- converts cytosine to uracil --> C-G to A-T

Question 31

# base editor dcas13 + cytosine deaminase

Answer 31

- converts adenosine to inosine (guanine) - corrects missense mutations in mRNA transcripts --> no dsDNA breaks

Question 32

# base editor hutchinson-gilford progeria syndrome (HGPS)

Answer 32

- fatal genetic disorder: rapid aging in kids, CVD, premature death - pathogenic C to T mutation --> splice site in LMNA --> mis-splicing --> truncated protein (toxic)

Question 33

# base editor CRISPR-STOP

Answer 33

makes stop codon --> premature stop --> knocked down gene

Question 34

# base editor CRISPR-SKIP

Answer 34

skips exon by changing into a different codon --> not used by machine

Question 35

cas13

Answer 35

cuts RNA, base changes, increases splicing

Question 36

prime editing

Answer 36

- includes template in sgRNA --> pegRNA includes primer binding site + cas9 + reverse transcriptase - reverses transcription --> new genetic info is written into target site - incorporate edit into target DNA

Question 37

split-cas9

Answer 37

regulate prime editing --> only active when these 2 are brought together

Question 38

live imaging of cellular genome

Answer 38

- studying complex chromosomal architecture and nuclear organization --> alternative to FISH - label dcas9 with fluorescent dye or add scaffold

Question 39

collateral cleavage activity

Answer 39

possible due to the many cas13 enzymes --> induce cleavage of nearby, non-target RNAs after cleavage of target sequence

Question 40

DETECTR

Answer 40

- DNA endonuclease-targeted CRISPR trans reporter - collateral cleavage activity of cas12a - nucleic acid detection system

Question 41

SHERLOCK

Answer 41

- specific high sensitivity enzymatic reporter unLOCKing - cas13a + sgRNA + fluorescent RNA reporters - after cas13 cuts target sequence, it cuts reporter RNA and releases detectable fluorescence for diagnosis