Source 1primary paper2022
Toolkit/stem-loop PEs
stem-loop PEs
Also known as: sPEs
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Stem-loop prime editors (sPEs) are modified prime editing guide RNA constructs in which stem-loop aptamers are added to the 3′ terminus of the pegRNA. This altered pegRNA architecture is used as a basis for tethered prime editors and for split pegRNA prime editor configurations.
Usefulness & Problems
Why this is useful
sPEs are useful because they directly address concerns that the 3′ extension of pegRNAs can impair pegRNA stability or folding and thereby compromise prime editing activity. The stem-loop architecture also provides a modular handle for building tethered and split prime editing systems.
Source:
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Problem solved
This construct pattern helps solve the problem that the 3′ extension of pegRNAs may negatively affect pegRNA stability or folding, reducing prime editing performance. It also addresses the engineering need for more flexible prime editor architectures, including tethered and split formats.
Problem links
Need conditional recombination or state switching
DerivedStem-loop prime editors (sPEs) are modified prime editing guide RNA constructs in which stem-loop aptamers are added to the 3′ terminus of the pegRNA. This altered pegRNA architecture is used as a basis for tethered prime editors and for split pegRNA prime editor configurations.
Need controllable genome or transcript editing
DerivedStem-loop prime editors (sPEs) are modified prime editing guide RNA constructs in which stem-loop aptamers are added to the 3′ terminus of the pegRNA. This altered pegRNA architecture is used as a basis for tethered prime editors and for split pegRNA prime editor configurations.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Techniques
No technique tags yet.
Target processes
editingrecombinationImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: regulatorswitch architecture: split
sPEs are constructed by adding stem-loop aptamers at the 3′ terminus of the pegRNA. The resulting modified pegRNAs can be tethered to Cas9 nickase to form tethered PEs, or split into an sgRNA and a prime RNA to create split pegRNA prime editors.
The supplied evidence does not provide quantitative editing outcomes, target scope, organismal validation, or direct comparative performance data for sPEs. Independent replication is not documented in the provided material, and the evidence is limited to a single 2022 source.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
Stem-loop PEs can be tethered to Cas9 nickase to produce tethered PEs.
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Stem-loop PEs can be tethered to Cas9 nickase to produce tethered PEs.
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Stem-loop PEs can be tethered to Cas9 nickase to produce tethered PEs.
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Stem-loop PEs can be tethered to Cas9 nickase to produce tethered PEs.
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Stem-loop PEs can be tethered to Cas9 nickase to produce tethered PEs.
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Stem-loop PEs can be tethered to Cas9 nickase to produce tethered PEs.
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Stem-loop PEs can be tethered to Cas9 nickase to produce tethered PEs.
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
The modified pegRNAs were split into sgRNA and prime RNA to create split pegRNA prime editors.
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
The modified pegRNAs were split into sgRNA and prime RNA to create split pegRNA prime editors.
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
The modified pegRNAs were split into sgRNA and prime RNA to create split pegRNA prime editors.
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
The modified pegRNAs were split into sgRNA and prime RNA to create split pegRNA prime editors.
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
The modified pegRNAs were split into sgRNA and prime RNA to create split pegRNA prime editors.
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
The modified pegRNAs were split into sgRNA and prime RNA to create split pegRNA prime editors.
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
The modified pegRNAs were split into sgRNA and prime RNA to create split pegRNA prime editors.
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
Split pegRNA prime editors maintain prime editing activity and increase flexibility.
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Split pegRNA prime editors maintain prime editing activity and increase flexibility.
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Split pegRNA prime editors maintain prime editing activity and increase flexibility.
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Split pegRNA prime editors maintain prime editing activity and increase flexibility.
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Split pegRNA prime editors maintain prime editing activity and increase flexibility.
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Split pegRNA prime editors maintain prime editing activity and increase flexibility.
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Split pegRNA prime editors maintain prime editing activity and increase flexibility.
The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
editing efficiency fold increase 2-4 fold
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
editing efficiency fold increase 2-4 fold
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
editing efficiency fold increase 2-4 fold
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
editing efficiency fold increase 2-4 fold
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
editing efficiency fold increase 2-4 fold
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
editing efficiency fold increase 2-4 fold
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
editing efficiency fold increase 2-4 fold
Approval Evidence
1 source3 linked approval claimsfirst-pass slug stem-loop-pes
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Source:
design rationalesupports
The 3-prime extension of pegRNAs could negatively affect pegRNA stability or folding and compromise prime editing activity.
The 3’-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity.
Source:
engineering strategysupports
Stem-loop PEs were generated by adding stem-loop aptamers at the 3-prime terminal of pegRNA.
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Source:
performance improvementsupports
Stem-loop PEs and tethered PEs increased small insertion, deletion, or point mutation editing efficiency by 2-fold to 4-fold on average in HEK293, U2OS, and HeLa cells.
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
Source:
Comparisons
Source-backed strengths
The design is explicitly modular, because the same stem-loop-modified pegRNA framework was used to generate both tethered PEs and split pegRNA prime editors. The available evidence supports a clear and simple engineering strategy: addition of 3′ stem-loop aptamers to pegRNA followed by optional tethering or splitting.
Source:
which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs)
Source:
Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3’-terminal of pegRNA
Source:
We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs)
Source:
sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells.
Compared with intron-containing CRISPRa construct
stem-loop PEs and intron-containing CRISPRa construct address a similar problem space because they share editing, recombination.
Shared frame: same top-level item type; shared target processes: editing, recombination
Compared with microfluidic organ-on-chip platforms
stem-loop PEs and microfluidic organ-on-chip platforms address a similar problem space because they share editing, recombination.
Shared frame: same top-level item type; shared target processes: editing, recombination
Strengths here: looks easier to implement in practice.
Compared with tethered PEs
stem-loop PEs and tethered PEs address a similar problem space because they share editing, recombination.
Shared frame: same top-level item type; shared target processes: editing, recombination; shared mechanisms: rna-protein tethering
Ranked Citations
- 1.