User:RenaMoonn/Workspace 4
From Wikipedia, the free encyclopedia
Prime editing is a 'search-and-replace' genome editing technology initially revealed in 2019. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. Prime editing allows for targeted insertions, deletions, and base-to-base conversions (both transitions and transversions) without the need for double stranded breaks (DSBs) or a donor DNA template.
History of development
If the thing didn't make a new editor part there's no need to include it here.
Initial concept
Prime editors were first conceptualized as a way to bypass the restrictions of traditional CRISPR-Cas9 gene editing as well as those of base editors.[1]
explain steps that were taken before developing prime editor 1
talk about how linker length was determined
Prime editor 1 (PE1)
Prime editor 1 was a prototype designed to have the minimum components necessary. It consisted of a wild-type Moloney murine leukemia virus (M-MLV) reverse transcriptase attached to Cas9 H840A nickase by a flexible linker. Originally, the linker was too short and the reverse transcriptase was unable to do its job. However, extending it to 8-15 bases led to detectable editing. During development, the linker was attached to either the C or the N terminal of the Cas9 protein. Linkage the C terminal proved more efficient, so it was chosen as the final design.[1]
look at extended data for efficiency stuff
Prime editor 2 (PE2)
After the success of PE1, researchers began modifying its reverse transcriptase. This was done to improve DNA synthesis and thus the editor's overall efficiency. They started by searching the literature for previously described mutations. These were found to improve the enzyme's thermostability, processivity, DNA to RNA substrate affinity, or prevent interference by RNaseH. A variety of mutations were combined into nineteen different variants and then tested against each other in human cells.[1]
Researchers began by focusing on thermal stability. They found that a reverse transcriptase combining D200N, L603W, and T330P mutations improved editing efficiency by an average of 6.8-fold for insertions and transversions at five different sites. Following this, the mutant transcriptase was further combined with additional mutations. Adding T306K and W313F improved editing efficiency by an additional 1.3 to 3-fold for insertions or transversions at the same five sites. The resulting prime editor was deemed PE2.[1]
Prime Editor 2 had a substantially higher efficiency than its predecessor when making insertion, deletion, or substitution mutations. Additionally, it was compatible with a shorter primer binding site (PBS) sequence due to its enhanced substrate affinity.[1]
Prime editor 3 (PE3)
PAM flexible PE2 and PE3 - 20 Nov 2020
study[2]
dunno yet - 9 April 2021
strange and obscure prime editors[3]
epegRNA - 4 Oct 2021
talk about epegRNA and how it helped with efficiency[4]
Prime editor 4 (PE4) - 28 Oct 2021
study[5]
Prime editor 5 (PE5) - 28 Oct 2021
study[5]
PEmax - 28 Oct 2021
enhanced prime editor architecture with improved efficiency, can be used with previous prime editor strategies to create PE2max, PE3max, PE4max, and PE5max[5]
Nuclease prime editor (PEn) - 24 Mar 2022
Upgraded nuclease prime editor (uPEn) - 19 Jan 2023
study[8]
Prime editor 6 - Aug 31 2023
there are multiple variants of these that can be used in different applications (a,b,c,d)[9]
Yeast-improved prime editor (PE_Y18) - 07 Mar 2024
study[10]
Prime editor 7 (PE7) - 03 Apr 2024
study[11]
Overview
General components
- Cas9 H840A nickase: talk about how Cas9 typically induces double stranded breaks using two different protein domains. One of them cleaves the target strand (explain what that is) and the other cleaves the non-target strand (also explain what that is). talk about how if you change part of the enzyme (explain what parts) one of the domain becomes nonfunctional. cas9 in prime editing is a H840A nickase which has mutation (explain what it is) that makes it so only the RuvC domain is functional. When this enzyme binds to DNA, only a single stranded break called a nick is. This is where the enzyme gets the name "nickase" from.
- Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase: tell where this protein is from. explain basics of how reverse transcriptase works, mention how it's fused to the prime editor with a linker protein and mention what terminal (part of the protein) the linker is located.
- pegRNA: modified version of a standard Cas9 guide RNA. explain the parts that are from typical Cas9 guide RNA and what aren't. cas9 binding sequence. guide sequence, Cas9 scaffold, reverse transcriptase template, primer binding site. RNA pseudo-knot improves efficiency (epegRNA)
Additional components
- MMR represser mRNA
- LA RNA protector protein
Editing mechanism
Original
NGG PAM. reverse transcriptase edits downstream of the cas9 protein, this because cas9 wraps around where it targets and makes editing that area inaccessible. cas9 cuts non-target strand 3 bp upstream of the PAM. stuff that was originally binded to guide sequence gets released and becomes primer site. primer binding region then binds to this, beginning the prime editing process. starting at the 5' end of the peg RNA (place right before the primer binding site, reverse transcriptase begins making DNA strand until it hits the scaffold. talk about incorporation process, yada yada yada
Paired prime editing - 28 Oct 2021
HOPE strategy: use two complimentary guide RNA pairs to increase chances of edit inclusion (just like Rin and Len guys!)[12]
Twin prime editing - 29 Dec 2021
pegRNA thing (spegRNA and apegRNA)[13]
PASTE - 24 Nov 2022
drag and drop using integrase[14]
PASSIGE - 10 Jun 2024
new strat that's way better than PASTE[15]
Delivery
might disregard this, but maybe avoid making this section a history thing and just use up-to date strategies. I guess if anything sounds cool you can include it
To use AAVs in mice, prime editor is split and then reassembled[16] PE6 has something to do with AAVs[9]
new engineered virus-like particles[17]
Applications
section doesn't need to be too big since there are genome editing in plants and animals thing. I'd focus the most on human genome therapy stuff because I'm a selfish human who doesn't care about other animals or corn
Cell cultures
Animal models
Efficient prime editing in mouse brain,liver and heart with dual AAVs[20]
zebrafish[21]
Agriculture
Ultra-efficient tomato prime editor[22]
rice[23]
Gene therapy
Section on prime editing therapy in humans that just released in 2025