Presentation 7: Plant Prime Editing Systems Flashcards
What is gene editing?
Improves the cultivar’s phenotype through precision mutation of its gene.
What is transgenic editing?
Improves a cultivar through incorporating an exogenous gene.
What is the issue with CRISPR when dealing with plants?
Poor understanding of plant Homology-directed repair
Double Strand Break (DSB)
• INDEL (34.1% rate)
• Off-target mutations (40% of on- target efficiency)
• p53 (tumour suppressor) activation = apoptosis
What is prime editing?
"Search & Replace" Searches for Single Strand Break (SSB)and can perform Transitions Transversions Insertions (up to 40bp) Deletions (up to 80bp)
What makes up the prime editor?
M MLV RT: Moloney Murine Leukemia virus RT
nCas9
pegRNA: Prime editing guide RNA (contains primer binding site, RT template, and guide RNA)
How does prime editing work?
1) pegRNA guides the nCas9 & M-MLV RT complex to the target sequence where it nicks before the target strand’s distal PAM sequence, exposing the 3’-hydroxyl group.
2) With the single strand nicked by nickase, the 5’ target strand leading to the distal PAM becomes extruded.
3) The exposed 5’ target strand gets hybridized to the pegRNA by the PBS, priming the 3’ Hydroxyl group for elongation by M-MLV RT.
4) Initiating at the 3’-Hydroxyl group of the extruded 5’ target strand, M- MLV RT elongates the edited DNA from pegRNA’s RT template.
5) Prior steps result in two extruded branched intermediates at the target site:
• 3’ Flap, contains the edited sequence
• Forms a heteroduplex with the Non-
target strand.
• 5’ Flap, the original unedited DNA sequence.
• Gets cleaved by either:
FEN1 :: 5’ - 3’ Flap Structure-Specific
Endonuclease 1
EXO1 :: 5’ exonuclease.
6) The 5’ Flap is cleaved by FEN1, followed by ligation.
• DNA mismatch repair (MMR) will resolve the heteroduplexes by copying the sequence of the edited target strand into the non-target strand or vice versa.
How did the researchers establish a prime-editing system for plants?
Generated a binary vector
H840A mutation introduced into SpCas9
What happened when the binary vector was created?
Replace HPT gene with HPT-ATG reporter
Insert artificial sequence in front of HPT-ATG
PeHPT1 will induce +1 C to T conversion
This mutation will generate a start codon for HPT recovering hygromycin resistance
What did PegRNA peWXI induce in the PE2 system?
PegRNA peWXI induces T to G transversion in OsWx gene (no mutations detected)
How was the PE3 Precise Genome Editing system developed?
Developed by nicking the unedited strand to increase the chance o fixing DNA mismatches according to the edited strands using an additional second gRNA
After the edited strand is synthesized, 5′ flap excision leaves behind a DNA heteroduplex
containing one edited strand and one non-edited strand.
The heteroduplex is resolved by mismatch repair to give either edited or non-edited products.
Nicking the non-edited strand causes repair of that strand, resulting in the generation of duplex DNA containing the desired edit.
What is the difference between PE3 and PE3b?
PE3: the second nick of the unedited strand is introduced away from the first nick site.
PE3b: the second nick is only introduced after the first nick is modified.
What happened with PE3?
PE3 on peACC1 at different nicking positions on target gene
• 1- bp deletion at the target of gRNA was detected in two plants at nicking position –31
Finally, there was no significant increase in mutant frequency observed in PE3/PE3b system
Which is Prime Editing better than CRISPR?
Prime Editing can serve as a powerful alternative for gene editing because it can perform transitions, transversions, insertions, and deletions.
No Indels in PE2 vs. CRISPR’s 34.1% rate.
Plant Prime Editor (pPE2): 0-31.3% efficiency vs. CRISPR’s 53-66%
Limited pPE2 efficiency as there were no homozygous mutations detected
Why may PE2 may not be as good as introducing deletions?
The PE2 system can be used to bring forth different types of mutations.
But in this experiment the 28 bp deletion provided 0 mutants. So, it may not be as efficient introducing deletions.
