Paired Nickases for Precise Polyploid Genome Editing
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Solution Overview
Problem
Current genome editing methods in alloploid and polyploid crops, such as wheat and cotton, face challenges in achieving precise gene edits due to high frequencies of random insertions and deletions (InDels) during DNA repair, requiring extensive screening to identify desired genotypes, and there is a need for efficient introduction of donor DNA into specific genome regions.
Innovation Solution
A method using paired RNA-guided nickases that create nicks in opposite strands of the DNA without inducing double-strand breaks, allowing for precise gene editing by facilitating homologous recombination when donor DNA with homologous overhangs is present, reducing the occurrence of InDels by maintaining hydrogen bonds between complementary bases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If double strand breaks are induced in the genome for genome editing, then gene editing capability is achieved, but random insertions and deletions (InDels) occur frequently during DNA repair
Solution Approach 1:
The invention divides the double strand break into two separate single strand nicks, introduced by two different Cas9 nickases targeting opposite strands at adjacent positions. This segmentation approach prevents the formation of double strand breaks, thereby avoiding error-prone NHEJ repair and reducing random InDels while maintaining gene editing capability through HR repair.
Solution Approach 2:
The invention introduces donor DNA as an intermediary molecule with homologous sequences that mediates the repair process. By providing a template for homologous recombination, the donor DNA guides precise repair of the nicked DNA, ensuring high precision gene editing without random mutations.
2Measurement precision
If extensive screening is performed to identify desired genotypes in alloploid and polyploid crops, then precise gene edits can be identified, but time and labor costs increase significantly
Solution Approach 1:
By segmenting the DNA break into two separate nicks on opposite strands, the invention creates a repair scenario that strongly favors homologous recombination over NHEJ. This results in predominantly precise edits with minimal InDels, dramatically reducing the number of plants that need to be screened to find desired genotypes in alloploid and polyploid crops.
3Reliability
If paired Cas9 nickases are used to induce double strand breaks, then off-target breaks are reduced, but the distance between nicks required for DSB induction is not clearly defined
Solution Approach 1:
The invention changes the critical parameter from nick distance (required for DSB induction) to nick absence (preventing DSB formation). By designing nickases that create single-strand nicks rather than double-strand breaks, the system achieves high specificity without requiring precise control of nick spacing, simplifying the design and reducing off-target effects.
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 significantly reduces the frequency of random mutations, enhancing the efficiency of precise gene editing in alloploid and polyploid crops by ensuring that DNA repair leads to either a wild-type or precise edit sequence, thereby minimizing the need for extensive screening and improving the accuracy of genome editing.
Implementation Method 1
the base pairs between the nicks would keep the complementary DNA strands together by keeping the hydrogen bonds between the complementary bases of the two strands intact
Data Source
AI summary
Genome editing including the introducing of precise gene edits is well established in diploid plants. Methods well established in the art introduce double strand DNA breaks in the genome of a plant applying technologies such as Zn-finger nucleases, homing endonucleases, TALEN or RNA guided nuclease e.g. Cas9 or Cas12a.