Double-Stranded DNA Break Repair Mechanisms

Nuclease-induced genome editing

1) use a guide RNA (sgRNA) to direct Cas9 to make a specific double-stranded DNA break within target sequence

2) when the cell detects the DSB, it will trigger DNA repair mechanism:

• non-homologous end joining (NHEJ): sticks broken ends back together

• homology-directed repair (HDR): using the homologous chromosome as template to repair the break and fill in any sequences that may be lost

Non-homologous end joining (NHEJ)

• results in small indels; can be used to create lof mutations (gene knockout)

• proteins that recognize and bind to the broken ends

• proteins that recruit and activate a nuclease, used to trim the broken ends

• the DNA ligase that connects the broken ends together

NHEJ (flip)

NHEJ Repair: Key Ideas

• after a DNA double-strand break, there may be loss of nucleotides from either strand as the ends unwind and degrade

• a complex of DNA binding protein kinases (the Ku proteins) recognize and bind to each broken end, recruiting a NHEJ polymerase, nuclease and ligases

  • this helps to connect the ends together and fill in any of the gaps

• the repaired DNA may have deletions or insertions as a result of the repair

• this form of repair can be used to direct loss of function mutations in target genes

Homology Directed Repair

• nucleases trim back the DNA to leave long 3’ tails

• recombination proteins bring one of the 3’ tails over to the homologous sequences of the other chromosome, and unwinds the other DNA so that the tail hybridize

• DNA polymerase can now extend the 3’ tail, using the other chromosome as a template

• once the newly synthesized DNA overlaps with the other 3’ tail, the repair complex dissociates and the two tails can hybridize

• DNA polymerase can fill in the gaps, and DNA ligase can connect the backbones

HDR (Flip)

HDR Continued (flip)

HDR: Key Ideas

• after a DSB, nucleases bind to the ends and degrade one strand to leave a long 3’ tail

• recombinase proteins allow the long 3’ ends to anneal to homologous sequences on the other chromosome. The homologous chromosome can then serve as the template as DNA polymerase further extends the 3’ tails

• eventually the 3’ tails are long enough for the ends to have complementary sequences, and they can anneal to each other. DNA polymerase fills in the gaps, and DNA ligase connects the phosphate backbone

• the repair would result in sequences that are copied from the template DNA (typically from the homologous chromosome)

• if you were to introduce a different template sequence, the repair could result in sequences copied from your engineered DNA template

C. elegans Gene Knockout: Large-scale approach to knocking out C. elegans genes using CRISPR technology

Design stage: Identify good target sequences to use in the guide RNA

• do the guide sequences have an optimal structure? (e.g. GC content, 3’ ends with GG, located next to PAM site within target”

• are they unique? eliminate any sequences that have multiple targets

• for KOs, generally best to target start of ORF

This became a database to help other researchers select guide RNAs for C. elegans research

Chop-Chop

• another tool to design sgRNAs

Why use a donor template to knock out a gene through homology-directed repair, when non-homologous end joining could also be used to create a loss-of-function mutation?

• NHEJ results in random changes to the sequence, whereas HDR changes would be specific and known

• ability to introduce selectable markers or reporter genes to help identify organisms with edited genomes

Design of the Donor Template

• recall that it is being used to disrupt targeted genes and we need an easy way to identify C. elegans that have beeb succesfully edited

  • note, in this study, 2 cuts were introduced in order to replace a longer stretch of sequences (requires 2 separate guide RNA constructs)

Design of the Donor Template 1

5’ homology arm and 3’ homology arm

• in order for a template to be used in HDR, it must be flanked by sequences that are homologous to the targeted area

• the homology arms should also cover the regions of the target sequence that will be cut

• in this case, the template is being used to replace target gene sequences with selectable marker genes

Design of the Donor Template 2

Selectable markers to detect transformed worms

• antibiotic resitance gene (neoR), to select for worms that have been edited sucessfully

• GFP used to track this mutation in crosses

  • a marker for having at least one deleted copy of the gene

  • fluorescence intensity reflects whether the worm is heterozygous or homozygous for the deletion (2x GFP)

  • Pmyo-2 and Prps-27 are promoters

Plasmids injected into C. elegans

• slide is showing the components you inject into C. elegans to carry out a CRISPR gene knockout (each plasmid has a specific role)

• need Cas9 plasmid with NLS signal to cut DNA

• need guide RNA to tell Cas9 where to cut

• need a repair template to disrupt the gene (used during DNA repair after Cas9 cuts)

Screening Process for Successfully Edited Worms