Prime Editing Flashcards
Primer editor components
- Cas9 protein
- Reverse transcriptase
- PegRNA (prime editing guide RNA): both use as a guide and template for desired editing
Prime editing process
See pic
Differentiation from other cas9 based editors
- Very versatile:
(1) Can address all 12 point mutations; deletion, insertion and inversion. Insertion with dual-flag can do replace and insertion edits to multiple hundreds of base pairs and deletions spanning thousands of DNA base pairs(2) Can edit many different cell types including post-Mitotic cells (conventional cas9-based editors need NHEJ and HDR in order to complete edits (which can only happen in actively dividing cells) - Potentially less Indels / off-target edits due to three required DNA Ave pairing events, see pic; studies have demonstrated >99% occurrence of either a precise edit at the target site or maintenance of the unmodified target DNA sequence (ie either a precise edit happens at the target site, or no edits happens anywhere); there has been little to no observation of off-target editing with prime editing
- PAM: Prime editing also has a wider editing window; prime editing is not limited to editing within a specific window around the PAM sequence
- Specially advantageous in correcting CNS and skeletomuscular repeat expansion disorders: can effectively removing aberrant requence stretches ranging ~1.2 to 1.6 kilobases
Limitations?
Primarily limited by tissue-specific delivery modality
Cellular determinants of prime editing efficiency?
Post-Mitotic cells
Post-mitotic cells are cells that have exited the cell cycle and no longer divide. This means that these cells have completed their final division and have permanently withdrawn from the cell cycle.
Examples of post-mitotic cells in the human body include neurons in the brain and skeletal muscle cells. These cells have a specialized structure and function that requires them to be stable and long-lived, and they do not typically undergo cell division. Other examples of post-mitotic cells include mature red blood cells and certain types of cells in the lens of the eye.
Why is primer needed in prime editing?
In prime editing, a primer is used to direct the insertion of new genetic information into a specific location in the DNA sequence. The primer is a short piece of RNA that is designed to bind to the target DNA sequence and direct the activity of the prime editing enzyme to the correct location.
The prime editing enzyme consists of two components: a reverse transcriptase that can generate a new DNA sequence from an RNA template, and a Cas9 protein that can cut the DNA at a specific location. The primer is designed to be complementary to the DNA sequence just upstream of the location where the new genetic information will be inserted.
When the primer binds to the target DNA sequence, it serves as a template for the reverse transcriptase component of the prime editing enzyme. The reverse transcriptase uses the primer as a template to generate a new DNA sequence that contains the desired genetic information.
The Cas9 component of the enzyme then cuts the DNA at a specific location downstream of the new genetic information, creating a nick in the DNA strand. This nick allows the new DNA sequence to be incorporated into the DNA strand by the cell’s natural DNA repair machinery.
What are the component of pegRNA?
The main components of a pegRNA include:
Scaffold: The scaffold is a synthetic RNA molecule that serves as a backbone for the pegRNA. It helps to stabilize the pegRNA and guide it to the target DNA site.
Primer binding site: The primer binding site is a short DNA sequence at the 3’ end of the pegRNA that is designed to bind to the target DNA site and provide a template for the reverse transcriptase activity of the prime editing enzyme.
Editing window: The editing window is a stretch of RNA nucleotides in the pegRNA that spans the site where the desired genetic changes will be made. The editing window is complementary to the target DNA sequence and provides a template for the reverse transcriptase activity of the prime editing enzyme. (Can be 1-2 kilo bP)
Nicking site: The nicking site is a short RNA sequence in the pegRNA that is complementary to the non-edited strand of the target DNA. It guides the Cas9 nickase component of the prime editing enzyme to the correct location and ensures that a single-strand break is made in the non-edited DNA strand, allowing the edited DNA sequence to be incorporated.
RNA stem-loop structure: The RNA stem-loop structure is a region in the pegRNA that helps to stabilize the molecule and protect it from degradation.
Special Capability
Can perform deletions, insertions and inversions of entire kilobase, DNA sequences and perform combinations of any of these edits.
Editing window
- The editing window refers to the range of genomic positions or sites that can be targeted and edited using a particular genome editing technique.
Different genome editing techniques have different editing windows based on their mechanism of action and the specificity of their targeting components. For example, traditional CRISPR-Cas9 editing requires a specific protospacer adjacent motif (PAM) sequence to be present near the target site in order for the Cas9 enzyme to bind and cut the DNA. This limits the editing window to specific genomic regions that have the appropriate PAM sequence nearby.
Other genome editing techniques, such as base editing and prime editing, have different editing windows based on their specific mechanisms. Base editors can only convert certain bases to other bases, limiting their editing window to specific nucleotides within the genome. Prime editing has a wider editing window than base editing because it can introduce precise changes to the DNA sequence without creating double-stranded breaks or relying on homologous recombination, allowing it to target a wider range of genomic positions
BE4 and BE5
PegRNAs can degrade in cells and resulting in truncated pegRNAs (Long string of RNA at the end of the pegRNA that encodes the edit was susceptible to degradation by cellular enzymes) — to overcome this challenge, a protective structures are added. The structure modifies the pegRNA into knot-shaped structures and prevent them from RNA-degrading enzymes (epeg RNA: engineered pegRNAs)
DNA mismatch repair process interferes with editing efficiency and increases the fraction of unintended insertions or deletions (During MMR, exonuclease will randomly remove NT and then add NT) — to overcome this challenge a protein: MLH1dn is added to PE4 and PE5 to temporarily inhibit one component of mismatch repair
PE4 and PE5 are designed to overcome these challenges
Deep dive read:
https://www.broadinstitute.org/news/next-generation-prime-editing-systems-move-closer-possible-therapeutic-applications
Why is prime editing PAM flexible?
Different variants of dCAS have been developed to target various different PAM sequences.
