Prime Editing Guide RNA for FANCC Gene Correction
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Solution Overview
Problem
Fanconi anemia of complementation group C (FA-C) patients face progressive bone marrow failure and cancer predisposition due to mutations in the FANCC gene, with current treatments being partially effective and involving sensitivity to DNA-damaging agents, necessitating a more precise genetic correction method.
Innovation Solution
Prime editing technology is employed to specifically target and correct mutations in the FANCC gene using a prime editing guide RNA (PEgRNA) that recognizes and edits the FANCC gene, introducing nucleotide edits to restore functional DNA repair pathways, thereby reducing sensitivity to DNA-damaging agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional treatments are used for Fanconi anemia, then patients receive standard care, but treatment effectiveness is limited and patients remain sensitive to DNA-damaging agents
Solution Approach 1:
The treatment approach is segmented into two distinct CRISPR-Cas9 systems: a first CRISPR-Cas9 system that introduces a premature stop codon to knock out the wild-type FANCC allele, and a second CRISPR-Cas9 system that corrects the mutant FANCC allele. This segmentation allows independent optimization of each editing event, improving overall treatment effectiveness while avoiding the sensitivity issue through precise allele-specific editing.
Solution Approach 2:
The patent employs preliminary action by first knocking out the wild-type FANCC allele using a CRISPR-Cas9 system with a guide RNA targeting the wild-type allele, before subsequently correcting the mutant allele. This preliminary knockout prevents potential harmful interactions and establishes a clean genetic background for the subsequent correction, thereby improving treatment reliability and reducing sensitivity to DNA-damaging agents.
2Reliability
If genome editing is used to correct FANCC mutations, then DNA repair pathways can be restored, but the complexity of the editing process increases
Solution Approach 1:
The complex genome editing process is divided into two separate, sequential CRISPR-Cas9 editing events. The first event knocks out the wild-type allele using a simplified approach with a guide RNA and donor template, while the second event corrects the mutant allele. This segmentation makes each individual editing step less complex and more controllable, while collectively achieving the restoration of DNA repair pathway function.
Solution Approach 2:
The patent uses an intermediary approach by employing a premature stop codon as an intermediate step. The first CRISPR-Cas9 system introduces this stop codon to knockout the wild-type allele, serving as an intermediate state that prevents harmful wild-type protein expression before the final correction of the mutant allele. This intermediary step simplifies the overall process by creating a clear intermediate milestone in the editing pathway.
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
The prime editing approach effectively corrects disease-causing mutations in the FANCC gene, potentially improving bone marrow function and reducing cancer risk in FA-C patients by enhancing DNA repair mechanisms, offering a more precise and targeted therapeutic option compared to existing treatments.
Implementation Method 1
the spacer sequence of a PEgRNA recognizes and anneals with a search target sequence in a target strand of the target gene
Implementation Method 2
A prime editing complex may generate a nick in the target gene on the edit strand which is the complementary strand of the target strand
Implementation Method 3
a single stranded DNA is synthesized using an editing template of the PEgRNA as a template. The editing template may comprise one or more nucleotide edits compared to the endogenous target FANCC gene sequence
Implementation Method 4
a gRNA core capable of binding to a Cas9 protein
Data Source
AI summary
Provided herein are compositions and methods of using prime editing systems comprising prime editors and prime editing guide RNAs for treatment of genetic disorders.