IAS04

Question 1

CRISPR & Cas9 names

Answer 1

clustered regularly interspaced short palindromic repeats
CRISPR-associated proteins: endonuclease involved in cutting DNA w/ molecular scissors

Question 2

bacterial CRISPR locus

Answer 2

CAS genes encode CAS proteins (cutting DNA)
Spacers same seq as phage DNA -> acquired bacterial immunity against phages

Question 3

CRISPR mechanism in bacteria

Answer 3

• phage DNA inserted into bacteria
• guide RNA & tracr RNA formed, guide RNA variable & comp. to phage DNA seq., tracr RNA constant, pair w/ guide RNA & scaffold w/ Cas9 to stabilize structure
• form complex w/ Cas9
• Cas9 finds PAM at 3’ end of target seq. -> binds to region of PAM -> match of guide RNA w/ phage DNA seq. -> if match, CUT

Question 4

adding DNA into CRISPR locus (OPTIONAL)

Answer 4

Cas1-Cas2 identify viral DNA -> cuts DNA sections next to PAM (protospacer) -> inserted in new locus of CRISPR array separated by palindromic repeats

Question 5

PAM (CRISPR)

Answer 5

protospacer adjacent motif
distinguish phage DNA w/ CRISPR locus as only phage DNA contains PAM
located just downstream of target sequence of DNA
search PAM before binding -> more efficient searching of target seq.

Question 6

CRISPR in lab

Answer 6

change guide RNA to cut DNA at any specific location, in vitro or in vivo
guide RNA & tracr RNA fused (by loop) -> sgRNA

Question 7

CRISPR applications (human)

Answer 7

gene silencing: after cut DNA repairs itself -> error prone -> mutations arise which disable gene -> elinimate mutant genes
gene replacement: cut end integrates w/ added DNA of desired seq., replacing original seq.
gene editing / gene therapy / agriculture

Question 8

CRISPR benefits

Answer 8

specificity & wide range of use

Question 9

dead Cas9 (dCas9)

Answer 9

cutting domains inactivated -> cannot cut but has binding abilities
- fluorescent proteins for easy observation
- change base (deaminase remove amine group, C->T)
- promote transcription by attaching deactivated Cas9 to transcription factors / activator proteins directly or via peptide
- inhibit transcription by recruiting factors to block off binding site for TF

Question 10

CRISPR ethical issues

Answer 10

unknown effect on babies, chromothripsis (shattering of chromosomes), genotoxicity, too risky to be used in germ cells now

Question 11

CRISPR clinical trials

Answer 11

LCA (leber congenital amaurosis), point mutation genetic disorder leads to blind
SCD & beta-thalassaemia: Casgevy