Cas9 Nickase Gene Conversion via 5' Overhangs
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Solution Overview
Problem
Current CRISPR/Cas systems face challenges in efficiently utilizing gene conversion pathways for genome editing, particularly in mammalian cells, where the efficiency of modifying target genes using endogenous homologous regions as donor templates is limited.
Innovation Solution
The use of two Cas9 nickases and guide RNAs to introduce specific single-strand breaks in a target gene, creating 5' overhangs that are repaired via gene conversion using an endogenous homologous region, thereby modifying the target gene without the need for exogenous nucleic acid templates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional CRISPR/Cas systems are used to create double-strand breaks, then genome editing can be achieved, but the efficiency of gene conversion using endogenous homologous regions is limited
Solution Approach 1:
The invention divides the single Cas9 double-strand break into two separate Cas9 nickase single-strand breaks, each guided by a separate gRNA. This segmentation allows the system to create a nick on one strand followed by a nick on the complementary strand, generating a clean double-strand break with 5' overhangs that are more favorable for gene conversion repair pathways.
Solution Approach 2:
The invention changes the cutting mode from a single Cas9 nuclease creating blunt or irregular ends to two Cas9 nickases creating controlled 5' overhangs. This parameter change in the break geometry promotes end resection and homology-directed repair, thereby increasing gene conversion efficiency while maintaining precision.
2Manufacturing precision
If exogenous donor templates are used for gene correction, then precise editing can be achieved, but the complexity of the system increases
Solution Approach 1:
The invention enables the cell to use its own endogenous homologous regions as donor templates for repair. Instead of requiring external delivery of donor DNA templates, the system leverages the cell's native genomic sequences that share homology with the target region, allowing the cell to self-service the repair process using available resources.
Solution Approach 2:
The invention makes the endogenous homologous regions serve dual functions: they remain as part of the normal genome structure and simultaneously function as donor templates for repair. This multi-functionality eliminates the need for separate donor template components, reducing system complexity while maintaining precision.
3Productivity
If Cas9 nickases are used to create single-strand breaks, then gene conversion efficiency is increased, but the number of components required increases
Solution Approach 1:
The invention combines two Cas9 nickase-gRNA complexes into a coordinated system where both components target the same genomic locus. By merging their functions to create a cooperative double-nick mechanism, the system achieves enhanced gene conversion efficiency while the components work together as an integrated unit rather than separate independent actions.
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 increases the rate of gene conversion-mediated modifications, allowing for precise correction of mutations in genes associated with diseases like sickle-cell anemia and beta-thalassemia, with repair efficiencies observed in a substantial percentage of cells, including hematopoietic stem cells.
Implementation Method 1
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.
Implementation Method 2
After the formation of a DNA double-stranded break (DSB), the major decision point affecting DNA repair pathway choice is whether or not the DNA ends are endo- and exonucleolytically processed in a process referred to as end resection. When no end resection takes places, the repair pathway engaged to repair the DSB is referred to as classical non-homologous end joining (C-NHEJ). In contrast, if the end resection machinery processes the DSB, a 3′ overhang is exposed, which engages in homology search.
Implementation Method 3
When the end resection is extensive, the exposed 3′ overhang can undergo strand invasion of highly homologous sequences, followed by repair of the DSB by a homology-dependent recombination (HDR) pathway, which encompasses gene correction and gene conversion. Gene correction refers to the process of repairing DNA damage by HDR using an exogenous nucleic acid, e.g., an exogenous donor template nucleic acid. Gene conversion is a process whereby a DNA break leads to repair of the sequence in the vicinity of the break by an endogenous homologous sequence, referred to as an endogenous donor template sequence.
Data Source
AI summary
CRISPR/CAS-related compositions and methods for altering a cell or treating a disease, for example, by gene conversion, are disclosed.