CRISPR

What is the only modification we can make?

Cut DNA

What are the two previous genome editing technologies? What is the major change to the new ones?

ZFN, Talen

Easier to program

What did Yoshizumi Ishino discover?

The repeat structure in bacteriophage

What does CRISPR stand for

Cluster regularly interspaced palindromic repeats

What was discovered in 2012

Cas9 cleaves target DNA with protospacer

What does Cas 9 stand for

CRISPR associated protein 9

Part of prokaryotic immune system that defends against viruses

What is the natural CRISPR pathway?

• DNA invasion

• Invading DNA is incorporated into CRISPR Array

• Pre crRNA transcription

• Guide RNA formation

• Cas9 activation

• Target binding

• Target cleavage

What are the 3 key elements?

• Cas9 DNA endonuclease

• a crRNA which contains a 20 bp sequence complementary to target

• Trans-activating crRNA (bridge between the crRNA and Cas9 enzyme

crRNA binds to the complementary DNA strand upstream of the PAM sequence

What is a possible unwanted situation

Off target cleavage- genome is huge so there will be similar sequences

crRNA spacer sequence

3’ end of the DNA must have PAM ( proto-spacer adjacent motif) sequence (5’NGG-3”), the 20 nucleotides upstream of the PAM sequence will be targeted, will cleave approximately three basses upstream of the PAM sequence

Not part of or included in the sgRNA

Which forms can it be delivered by?

DNA, mRNA, protein

What are some methods of delivery?

• Viral AAV (for muscular dys.)

• physical- microinjection or electroporation

What is dCas

Deactivated cas 9- just changes 1 base pair, still capable of changes just less side effects

What are the three required elements of base editing?

• A Cas nickase or Cas fused to deaminase that makes the edit

• A g RNA targeting Cas to a specific locus

• A target base for editing within the editing window specified by the cas protein

CRISPRa

Increases expression complex, CRISPRi does opposite

In vivo vs ex vivo

In- eye, ear

Ex- blood dieases, sickle cell anemia, hemophilia, CAR T cancer

Somatic vs Germline

Somatic- not reproductive cells, less complicated

Technologies

• PRIME editing- reverse transcriptase fused to Cas9 nickase and a prime-editing guide RNA (pegRNA)

• Base editing

• CRISPERa/i- dCas

• Epigenome editing- p300 protein, relax complex

• Screening

What is the PAM sequence?

TGG

What species was the CRISPR system first discovered in?

E.Coli