DSB Repair and Genome Editing

Question 1

Sources of double strand DNA breaks

Answer 1

• Ionizing radiation and reactive oxygen species
• Type 2 topoisomerases cleave and reseal DNA but if resealing fails -> DSB
• DSBs facilitate recombination between homologous chromosomes
• If replication fork encounters single strand break, lack of non covalent interactions cause replication arm to separate from fork resulting in DSB
• If DSBs persisted throughout mitosis or meiosis, lead to detrimental loss of genetic information

Question 2

Homology Directed Repair

Answer 2

• HDR happens if resection occurs: process where nucleases trim back broken DNA ends to create single stranded overhangs
• Sub pathways: Single Strand Annealing (SSA) and Homologous recombination
• If ssDNA strands are homologous, follow SSA pathway: complementary strands base pair together, undergo flap cleavage, gap filling, and ligation to repair DSB
• If ssDNA overhangs not complementary, homologous recombination occurs through more complex multi step process involving strand invasion, double holliday junction (dHj) formation, and resolution

Question 3

Non homologous end joining

Answer 3

• Happens when resection does not occur
• Ends of DSB can interact through perfect synapsis or incorrect microhomology
• Perfect synapsis: broken strands reanneal and are ligated (no change in sequence)
• Microhomology: Incorrect base pairing occurs leading to addition and deletion of nucleotides

Question 4

Homologous recombination - end resection

Answer 4

• MRN outcompetes Ku for DSB binding -> recruit CtIP
• MRN + CtIP complex begins 5’-3’ resection of DNA generating 3’ ssDNA overhangs -> used to locate repair template and bound by RPA for stabilization
• Complex removes 100 nucleotides as initiation
• Resection continues using exonuclease Exo1, resect DNA at rate of 4 kilobases per hour

Question 5

Homologous recombination - strand invasion

Answer 5

• Suitable repair template identified by RAD51 and ssDNA. After end resection, ssDNA coated with RPA to prevent RAD51 binding. BRCA2 exchange RPA with RAD51 and ssDNA stabilized by RAD51 paralogs
• RAD51 looks for any strand that can base pair with 3’ssDNA overhang
• RAD51 inserts itself into double helix of dsDNA repair template to catalyze base pairing between RAD51 filament and complementary strand of repair template. Referred to as strand invasion and forms D loop
• Produces two primed template for DNA polymerase. RAD51 must be removed by RAD54 so replisome can access template. Formation of double Holliday junction
• ## Nuclease cleavage to resolve dHj. Cause recombination between template and previously broken DNA

Question 6

Potential Repair Templates

Answer 6

• Sister chromatid because it contain same nucleotide sequence as original DNA prior to DSB (most preferred)
• Homologous chromosome because nucleotide encodes same genes
• Retrotransposon but this can lead to detrimental effect such as chromosomal translocation and rearrangement
• Can bind to artificially introduced template

Question 7

Experimental evidence for BRCA2

Answer 7

• Four substrates: ssDNA overhang, repair template, RAD51, BRCA2 in various concentrations
• If successful strand invasion: Band near top of gel with intensity correlating with efficiency of reaction at each concentration of BRCA2
• If unsuccessful: Band near bottom of gel
• Negative control: band at bottom of gel, positive control: band at top of gel
• Results: As BRCA2 concentration increased, band appear at top of gel

Question 8

Non-homologous end joining

Answer 8

• Ku outcompete MRN, Ku acts as scaffold and ends of broken DNA brought back together
• ## DSBs asymetrical: break at slightly different locations

Question 9

Outcomes of NHEJ

Answer 9

• No sequence change: ends reanneal in same manner prior to being broken
• Insertion: Ends reanneal between first base pairs in single stranded DNA. result in insertion of new bases
• Deletion: ends reanneal between bases near end of single stranded DNA segment. results in deletion of overhanging bases

Question 10

Initial attempt at genome engineering

Answer 10

• Introduce recombinant DNA molecule containing desired gene for integration and flanking sequences into yeast cell
• Theoretically artificially introduced DNA molecule perceived as DSB triggering initiation of DSB repair leading to recombination between recombinant DNA molecule and cellular chromosome
• Yeast preferentially undergo homologous recombination instead of NHEJ -> low efficiency

Question 11

Improved attempts of genome editing

Answer 11

• More efficient if double stranded break also introduced at target chromosmal locus
• Allowed for end resection to occur on both artificial and chromosomal DNA to create complementary 3’ ss overhangs that could anneal to each other
• Single Strand Annealing (SSA): Very few enzyme mediated steps including flap processing and ligation, more efficient

Question 12

CRISPR Cas9 mechanism of action

Answer 12

• Main components: Cas9 protein, 20 nucleotide crRNA, and structural tracrRNA
• Cas9 makes complex with cRNA and tracrRNA -> bind to DNA and scan for three nucleotide Protospacer Adjacent Motif (PAM)
• Identification of PAM leads to unwinding of dsDNA, checked for complimentarity against bound cRNA sequence, if complementary Cas9 induces double stranded break in DNA and dissociate
• cRNA bound with tracrRNA forms sgRNA. When no sgRNA bound, Cas9 in inactive apo state.

Question 13

Transition from inactive apo Cas9 to active

Answer 13

• Binding of sgRNA to Cas9 to scan for PAM sequences
• Cas9 + sgRNA scans for 5’-NGG-3’ PAM sequences. Cas9 complex dissociates from non PAM DNA
• Once PAM is encountered, second conformational change to unwind dsDNA. DNA checked for is on opposite side of PAM, referred to as target strand
• If target strand complementary to sgRNA, they anneal and third conformational change occurs. Cleave two genomic DNA strands using endonuclease domains RuvC and HNH, cleaving 3 nucleotides upstream of PAM. Non target strand cleave by RuvC and targets strand cleave by HNH

Question 14

Repair Mechanisms after CRISPR Cas9 Cleavage

Answer 14

• Change in nucleotide sequence generates a knockout: Frameshift mutation may occur through insertion or deletion of nucleotides
• Change in nucleotide sequence doesn’t generate knockout: Addition or deletion does not significantly alter product. If three amino acids added by NHEJ, single amino acid with minimal impact on function
• No change in nucleotide sequence: Correct rejoining occurs without addition and deletion of nucleotides, restoring original DNA sequence. Cas9 can bind again
• If DSB repaired by HDR, desirable genes can be integrate -> can be used to study gain of function proteins

Question 15

Use of CRISPR Cas9

Answer 15

• HIV Prevention: CCR5 is T cell receptor that HIV uses to gain entry into cell. naturally occurring 32 base pair deletion confers HIV resistance. Use a delta32 stem cell transplant to confer resistance in hosts
• Germline edits: Not accompanied using repair template, use NHEJ to generate random genetic variants to result in CCR5 knockout. Made at germline level