Engineered Cas9 Systems for Expanded Genome Coverage
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Solution Overview
Problem
Current CRISPR/Cas systems are limited by the requirement for specific protospacer adjacent motifs (PAMs), restricting genome coverage density and precision in genome editing, especially in complex eukaryotic genomes.
Innovation Solution
Development of engineered Cas9 systems that utilize alternate PAMs, including A and/or T residues and GC-rich sequences, to increase genome coverage density and enable targeted genome editing of previously inaccessible genomic loci.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If SpyCas9 with 5'-NGG-3' PAM is used for genome editing, then the system is widely adopted and relatively simple to use, but it is excluded from many genomic sites lacking such a motif
Solution Approach 1:
The patent changes the PAM sequence parameter from the conventional 5'-NGG-3' to alternative PAM sequences (5'-NAG-3', 5'-AAC-3', 5'-AAT-3'). This parameter change allows the Cas9 system to recognize and bind to previously inaccessible genomic sites, thereby expanding genome coverage while maintaining the fundamental CRISPR/Cas9 editing mechanism
Solution Approach 2:
The patent creates a multi-functional Cas9 system that can operate with different PAM sequences. By engineering Cas9 variants that recognize multiple PAM types, the system achieves universality across diverse genomic regions, allowing a single platform to address both GC-rich and AT-rich genomic sites that were previously inaccessible
2Reliability
If CRISPR/Cas systems require specific PAM sequences for target DNA binding, then the system maintains specificity, but genome coverage density is restricted
Solution Approach 1:
The patent modifies the PAM sequence parameter to include alternative sequences with different nucleotide compositions (GC-rich vs. AT-rich). This enables the system to maintain high targeting specificity through precise PAM recognition while significantly increasing genome coverage density by accessing previously excluded genomic regions
Solution Approach 2:
The patent segments the genomic target space into different PAM-type regions (GC-rich and AT-rich). By developing Cas9 variants for each segment type, the overall genome coverage is divided and conquered, allowing comprehensive editing across heterogeneous genomic landscapes
3Manufacturing precision
If precision gene editing using HDR or base editors is performed, then accurate single base pair editing is achieved, but it requires a precise DNA binding position that limits applicable sites
Solution Approach 1:
The patent changes the PAM sequence parameter to provide flexible positioning options for precision editing. Alternative PAM sequences enable base editors and HDR systems to be positioned at various distances from the target site, maintaining precise single-base-pair editing capability while increasing targeting flexibility across different genomic contexts
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 engineered Cas9 systems achieve enhanced genome editing efficiency and increased targeting resolution, allowing for precise modification of chromosomal sequences in eukaryotic cells.
Implementation Method 1
each engineered guide RNA comprises a 5' guide sequence designed to hybridize with a target sequence in a double-stranded sequence
Data Source
AI summary
Engineered Cas9 systems that utilize alternate protospacer adjacent motifs for target DNA binding, nucleic acids encoding the engineered Cas9 systems, and methods of using the engineered Cas9 systems for modifying target chromosomal sequences in eukaryotic cells.