CRISPR Nickase Ligation for Precise Gene Editing
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Solution Overview
Problem
Current CRISPR-based gene editing technologies face challenges such as off-target editing, chromosomal rearrangements, and unintended indel formation, which compromise safety and efficacy for clinical applications.
Innovation Solution
The method employs a CRISPR nickase to cleave genomic DNA near a target site, utilizing a CRISPR guide RNA, a splint oligonucleotide, and a ligand oligonucleotide to incorporate a nucleic acid sequence into the genome through ligation, minimizing the risks of double-strand breaks and associated rearrangements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR wild type Cas9 system is used to produce double-strand breaks for gene editing, then editing efficiency is improved, but chromosomal rearrangements and off-target effects increase
Solution Approach 1:
The patent divides the double-strand break function into two separate single-strand nicking events using two different nickase variants (e.g., Cas9H840A and Cas9D10A) with different PAM requirements. Each nickase targets a specific strand at a different location, and the nicks are processed by cellular repair mechanisms to achieve editing without forming dangerous double-strand breaks. This segmentation resolves the contradiction by maintaining editing efficiency while eliminating chromosomal rearrangements.
Solution Approach 2:
The patent introduces a third-component oligonucleotide as an intermediary that facilitates precise editing at the target site. This oligonucleotide serves as a template for homology-directed repair, guiding the cellular repair machinery to incorporate the desired genetic changes without relying on error-prone non-homologous end joining. The intermediary oligonucleotide enables precise editing while avoiding off-target effects and chromosomal rearrangements.
2Object-affected harmful factors
If CRISPR nickase is used to avoid double-strand breaks, then chromosomal rearrangements are reduced, but editing precision and HDR efficiency decrease
Solution Approach 1:
The patent combines multiple nickase variants (e.g., Cas9H840A and Cas9D10A) with different PAM requirements into a single editing system. By merging these variants with different guide RNAs, the system creates coordinated single-strand nicks on opposite strands at different locations, which together enable precise editing through homology-directed repair without forming double-strand breaks. This merging approach maintains editing precision while avoiding chromosomal rearrangements.
Solution Approach 2:
The patent performs preliminary nicking of both DNA strands at specific locations flanking the target site before the actual editing occurs. These preliminary nicks create controlled single-strand breaks that are then repaired using a provided oligonucleotide template, ensuring precise editing at the intended location. This preliminary action on both strands enhances editing precision while maintaining the safety benefits of avoiding double-strand breaks.
3Object-affected harmful factors
If base-editing is used to avoid indels and chromosomal rearrangements, then safety is improved, but the ability to incorporate modified or non-canonical bases is limited
Solution Approach 1:
The patent uses a third-component oligonucleotide that serves as a template or copy for the desired genetic edit. This oligonucleotide contains the precise sequence changes including modified or non-canonical bases, and the cellular homology-directed repair machinery copies this template into the genome at the target site. This copying approach enables incorporation of diverse base modifications while avoiding the safety issues of base-editing enzymes.
Solution Approach 2:
The patent creates a universal editing platform that can incorporate any desired sequence change by simply changing the oligonucleotide template, without requiring different enzymatic systems for different types of edits. The nickase-based HDR system is multi-functional and can accommodate canonical and non-canonical bases, modified bases, and various sequence modifications through a single unified mechanism, providing greater versatility than base-editing.
4Productivity
If double-strand break repair via non-homologous end joining is used, then gene knockout efficiency is improved, but unintended indel formation increases
Solution Approach 1:
The patent inverts the conventional approach by using single-strand nicks instead of double-strand breaks, and by providing a precise oligonucleotide template instead of relying on error-prone non-homologous end joining. This inversion of the repair mechanism switches from the imprecise NHEJ pathway to the precise homology-directed repair pathway, achieving accurate editing while maintaining gene disruption capability when needed.
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
This approach enhances the precision and safety of gene editing by reducing off-target effects and unintended genomic alterations, improving the flexibility and reliability of gene editing processes.
Implementation Method 1
a CRISPR guide RNA that includes a guide sequence that hybridizes with the target strand of the double-stranded nucleic acid
Implementation Method 2
the CRISPR guide RNA and Cas nickase form a complex with the double-stranded nucleic acid and make a single-strand cut in the displaced strand to form a nick in the displaced strand
Implementation Method 3
the splint oligonucleotide hybridizes to the displaced strand adjacent to the nick
Implementation Method 4
ligating one end of the ligand oligonucleotide to a nucleotide adjacent to the nick in the displaced strand to thereby result in the ligand oligonucleotide to be incorporated into the double-stranded nucleic acid
Data Source
AI summary
Disclosed are compositions and methods for gene editing. The present disclosure relates to compositions and methods for gene editing using a Cas nickase to cleave a double-stranded nucleic acid sequence near a target site and a ligase to incorporate a nucleic acid into a double-stranded nucleic acid sequence. The present disclosure also provides reagents for use in the gene editing methods. The present disclosure further provides kits containing reagents for use in the gene editing methods.