CRISPR/Cas9

Question 1

tracrRNA (3)

Answer 1

• trans-activating RNA
• region of complementarity with CRISPR repeat sequences
• works with crRNA

Question 2

cas9

Answer 2

• codes for a nuclease that cuts DNA with the same sequences as the spaces

Question 3

cas1, cas2, csn2

Answer 3

• codes for proteins that insert new sequences from invading DNA

Question 4

CRISPR array (2)

Answer 4

• contains repeat sequences separated by spacer sequences take from bacteriophages and other infectious elements
• transcribed to make the pre-crRNA (the pre-CRISPR RNA)

Question 5

how does CRISPR/Cas9 work in immunity: upon infection (3)

Answer 5

1. Cas protein complex samples infecting DNA
2. DNA is integrated as a new spacer in the CRISPR array between PAM sequences and tends to be behind leader sequence
3. cell and progeny have no adapted immunity against the invader

Question 6

how does CRISPR/Cas9 work in immunity: upon reinfection (4)

Answer 6

1. tracrRNA and pre-crRNA transcripts are made
2. two RNAs hybridize and are processed to shorter forms by Cas9 and RNase III
3. Cas9/crRNA/tracrRNA complex scans DNA for matches to its spacer sequence and the PAM sequence
4. Cas9 makes a ds blunt cut 3NT upstream of PAM site

Question 7

how does CRISR-Cas9 promote gene editing? (2)

Answer 7

• ability to direct dsDNA breaks at specific locations

- once the dsDNA break occurs, DNA repair systems take over and can be exploited to edit sequences

Question 8

DNA repair systems (2)

Answer 8

1. non-homologous end joining (NHEJ)

2. homology directed repair (HDR)

Question 9

how has the CRISPR/Cas9 system been modified to make targeted cuts within DNA?

Answer 9

• tracrRNA and crRNA sequences fuse to make single guide RNA (sgRNA) with combined function

Question 10

nuclease-induced genome editing via Non-homologous End Joining (NHEJ) (2)

Answer 10

1. nucleotides around ds break degrade and are lost
2. DNA binding protein kinases recognize and bind broken ends, recruiting NHEJ polymerase, nuclease, and ligases to connect ends together and fill in gaps

Question 11

nuclease-induced genome editing via Homology Directed Repair (HDR) (5)

Answer 11

1. ds break occurs and degradation occurs
2. nuclease binds to ends and degrades one strand, leaving a long 3’ tail on each side
3. recombination proteins brine one 3’ tail over to homologous sequences found in other chromosome; target DNA unwinds so 3’ tail can hybridze
4. DNA polymerase can add NTs to the 3’ end, using the target DNA as a template, until it reaches dsDNA again
5. newly synthesized end can hybridize to other 3’ tail: DNA polymerase can fill in the gap and DNA ligase can connect the backbones

Question 12

how is NHEJ unique

Answer 12

resulting repaired DNA may have deletions, insertions, or both (INDEL) after repair

Question 13

how is HDR unique?

Answer 13

• repaired strand has same sequence from before unless homologous strand used as a template has some differences

Question 14

after CRISPR/Cas9 is engineered to introduce precise dsDNA breaks, what can NHEJ do in gene editing?

Answer 14

• useful for knocking out gene function through deletions or insertios

Question 15

after CRISPR/Cas9 is engineered to introduce precise dsDNA breaks, what can HDR do in gene editing?

Answer 15

• ability to introduce specific mutations by supplying a template with the desired sequences

Question 16

when planning to knock-out genes, what is a critical part of the design stage? (2)

Answer 16

• identifying good target sequences to use in guide RNA: must be located next to PAM sites and be unique
• databases available to help