W4 CRISPR and Cas9 System

Question 1

what is the use of CRISPR-Cas systems

Answer 1

microbial adaptive immune system

protects bacteria against invading foreign material from viruses

Question 2

what is CRISPR

Answer 2

rna-guided nuclease that requires RNA to guide it to the genome to be cleaved

Question 3

definition of genetic memory

Answer 3

carries specific sequences to guide cas9 protein to target foreign dna

Question 4

key components of CRISPR-cas9 system

Answer 4

cluster of CRISPR-associated (Cas) genes, noncoding rans and array of repetitive elements

Question 5

what class and type does cas9 belong to

Answer 5

class 2 type II

Question 6

what are the three stages of CRISPR-Cas9 system

Answer 6

1. adaptation
2. expression of CRISPR array and processing (maturation of CRISPR-Cas9 complex)
3. interference: target recognition and target cleavage

Question 7

what are the 2 components of CRISPR RNA array

Answer 7

1. short repetitive sequences (25-40bp)
2. short variable sequences derived from exogenous dna targets

Question 8

what happens in stage 1

Answer 8

CRISPR system stores molecular signature of a previous infection

Question 9

what happens in stage 2

Answer 9

1. CRIPSR array is transcribed into pre-crRNA
2. pre-crRNA is processed into short crRNAs y cleavage in repeat sequences
3. TracrRNA facilitates processing of crRNA and maturation of tracrRNA-crRNA-Cas9 complex

Question 10

what happens in stage 3

Answer 10

1. crRNAs function as guide for cas9
2. each mature complex recognises and binds to target dsDNA sequence (foreign DNA complementary to crRNA) and cuts both strands

Question 11

what is the requirement for target dna recognition and subsequent cleavage by crRNA-tracrRNA-Cas9 complex

Answer 11

1. sequence complementary between the spacer and target dna sequence
2. presence of an appropriate potospacer-adjacent motif (PAM) sequence at 3’ end of target sequence

Question 12

what is the purpose of PAM recognition

Answer 12

1. facilitates Cas interference complex by binding and DNA melting
2. RNA:DNA heteroduplex formation
3. prevents self targeting of similiar/identical sequences lacking a PAM (prevents self cleavage)

Question 13

which components of the type II effector system are essential for CRISPR-Cas9 interference

Answer 13

guide rnas

tracrRNA

Cas9

Question 14

components important for type II CRISPR-Cas in genome editing

Answer 14

1. transcription of the locus yields a pre crRNA > processed to crRNAs consisting of spacer repeat fragments that guide effector nuclease complexes
2. crRNA array encodes the guide RNAs
3. tracrRNA mediates pre-crRNA processing and formation of cas9 complex
4. cas9 protein is the RNA-guided nuclease that mediates a dsDNA break (3bp upstream of PAM)

Question 15

how is functional crRNA-tracrRNA-Cas9 unit resonstitued to be used for mammalian cells

Answer 15

1. expression of a human-codon-optimized cas9 protein with an appropriate nuclear localisation signal
2. crRNA and tracrRNA are expressed either individually or as a single chimera via a RNA polymerase III promoter

Question 16

how can cas9-induced DSBs be repaired

Answer 16

1. non homologous en joining: error prone; re ligation leads to indel mutations > gene knockout
2. homology directed repair: high fidelity, more variable and less frequent process to generate specific point mutations/insertions at target locus > precise gene editing

Question 17

what is the repair template in repairing DSBs

Answer 17

conventional double stranded dna targeting plasmids with homology arms flanking insertion sequences

or

single stranded dna oligonucleotides

Question 18

what can sgRNA-directed cas9 nuclease induce

Answer 18

1. indel mutations
2. sequence specific insertion or replacement
3. sequence specific deletion