Multi-Flap Prime Editing for Precise Two-Strand DNA Editing
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Solution Overview
Problem
Current genome editing technologies face challenges in achieving precise and efficient single nucleotide changes, particularly in non-dividing human cells, with issues such as low efficiency of homology directed repair, generation of chromosomal rearrangements, and limitations in base editors like bystander editing and target nucleotide product mixtures.
Innovation Solution
The development of multi-flap prime editing systems, which involve generating pairs of 3' flaps on different DNA strands that form duplexes and are incorporated into the target nucleic acid, allowing for precise editing without double-strand breaks, using Cas9 nickase-reverse transcriptase fusions and prime editing guide RNAs to introduce desired edits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homology directed repair (HDR) is used for precise nucleotide editing, then editing precision is improved, but editing efficiency deteriorates (particularly in non-dividing cells)
Solution Approach 1:
The invention divides the editing process into two separate strands: a primer strand that initiates repair and a template strand that provides the edited sequence. This segmentation allows the system to bypass the inefficiency of traditional HDR in non-dividing cells while maintaining precision through the template-directed repair mechanism.
Solution Approach 2:
The invention introduces a single-stranded DNA primer as an intermediary molecule that bridges the gap between the double-strand break and the desired edit. This primer serves as a mediator that directs the cell's repair machinery to incorporate the edited template sequence, thereby improving efficiency without sacrificing precision.
2Productivity
If base editors are used for nucleotide editing, then editing efficiency is improved, but precision deteriorates (due to bystander editing and product mixtures)
Solution Approach 1:
The invention segments the editing function into separate components: the Cas9 nickase provides targeted binding and single-strand nicking, while the engineered reverse transcriptase performs the actual base conversion. This segmentation allows precise control over which bases are edited, eliminating bystander editing effects.
Solution Approach 2:
The invention applies local quality by designing the guide RNA to target a specific 3-nt window within the PAM-proximal region. This localized targeting ensures that only the desired nucleotide is edited, preventing off-target or bystander editing while maintaining high efficiency through the base editor mechanism.
3Adaptability or versatility
If CRISPR/Cas systems are used for genome editing, then versatility is improved (ability to access any genomic site), but harmful factors increase (chromosomal rearrangements and deletions)
Solution Approach 1:
Instead of using Cas9 to create a double-strand break and then relying on error-prone repair mechanisms, the invention inverts the approach by using Cas9 nickase to create only a single-strand nick. This inverted strategy avoids the harmful effects of double-strand breaks while still enabling precise editing through the primer-template mechanism.
Solution Approach 2:
The invention converts the potential harm of double-strand breaks into a benefit by using single-strand nicks, which are much less problematic for genomic stability. The single-strand nick is sufficient to initiate repair while avoiding the chromosomal rearrangements and deletions associated with double-strand breaks, thereby turning a weaker cutting mechanism into an advantageous feature.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables high-efficiency, flexible, and specific editing of nucleotides, including insertions and deletions, across various cell types, overcoming limitations of traditional methods by ensuring both strands of the DNA are edited simultaneously, thus enhancing therapeutic potential.
Implementation Method 1
a reverse transcriptase and wherein the guide RNA comprises a reverse transcription template sequence
Implementation Method 2
polymerizing a strand of DNA from the 3′ end, thereby generating a single-strand DNA flap comprising the desired nucleotide change
Implementation Method 3
nicking the double-stranded DNA sequence on the non-target strand, thereby generating a free single-strand DNA having a 3′ end
Implementation Method 4
the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop
Implementation Method 5
hybridizing the 3′ end of the free single-strand DNA to the reverse transcription template sequence, thereby priming the reverse transcriptase domain
Data Source
AI summary
The present disclosure provides systems, compositions, and methods for simultaneously editing both strands of a double-stranded DNA sequence at a target site to be edited. Further provided herein are pharmaceutical compositions, polynucleotides, vectors, cells, and kits for simultaneously editing both strands of a double-stranded DNA sequence.