Engineered Cas12b Base Editing for High-Specificity DNA Targeting
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Solution Overview
Problem
Current genome-editing technologies, such as CRISPR-Cas systems, face challenges in achieving high specificity, efficacy, and safety due to genetic variation in patient populations and off-target effects, necessitating the development of novel strategies for precise genome editing.
Innovation Solution
The use of engineered CRISPR-Cas effector proteins, particularly C2c1, with modifications to enhance binding and editing preferences, and delivery systems like lipid and viral particles for targeted DNA base editing, allowing integration of DNA inserts into eukaryotic genomes through non-homologous end joining and homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas systems are used for genome editing, then genome editing capability is achieved, but off-target effects occur reducing specificity
Solution Approach 1:
The patent applies local quality by engineering specific modifications to the Cas12b protein at particular amino acid positions (e.g., K870Q, R1005Q, R1068Q) to enhance binding affinity and specificity at the target site while maintaining overall protein function. This localized modification approach improves on-target editing precision without compromising genome editing capability.
Solution Approach 2:
The patent employs parameter changes by systematically varying guide RNA lengths (17-25 nucleotides), optimizing PAM sequence requirements (TTN, ATTN, TCTN), and adjusting Cas12b protein sequence parameters to achieve optimal specificity and activity. These parameter optimizations reduce off-target effects while preserving editing efficiency.
2Reliability
If engineered CRISPR-Cas effector proteins with modifications are used, then binding affinity and editing preference are enhanced, but protein complexity increases
Solution Approach 1:
The patent segments the Cas12b protein into functional domains and applies targeted mutations at specific positions (e.g., positions 870, 1005, 1068, 1177, 1191, 1207) rather than modifying the entire protein. This segmented approach allows precise control over binding affinity and editing preferences while minimizing overall protein complexity and maintaining structural integrity.
Solution Approach 2:
The patent creates composite effector proteins by fusing Cas12b with heterologous domains such as deaminases (e.g., APOBEC1, AID) to enable base editing functionality. This composite approach enhances editing capability and specificity while the modular design allows independent optimization of each domain's function.
3Productivity
If delivery systems like lipid and viral particles are used, then delivery efficiency is improved, but delivery system complexity increases
Solution Approach 1:
The patent develops universal delivery systems that can accommodate different CRISPR components (Cas12b protein, guide RNA, donor DNA templates) and target various cell types including non-dividing cells. The lipid nanoparticle and viral vector platforms are designed with modular architectures that can deliver multiple payloads simultaneously, improving delivery efficiency without requiring separate complex systems for each component.
Solution Approach 2:
The patent employs nested delivery structures where guide RNA and donor DNA templates are packaged within viral capsids or lipid nanoparticles that also contain the Cas12b protein. This nested arrangement protects all components during delivery while maintaining their individual functionalities, achieving high delivery efficiency through a single integrated delivery system rather than multiple separate administrations.
4Adaptability or versatility
If precise genome editing in non-dividing cells is achieved, then therapeutic applicability is improved, but editing efficiency decreases
Solution Approach 1:
The patent replaces the mechanical DNA double-strand break repair mechanism (which requires cell division for homologous recombination) with a chemical base modification mechanism using deaminase enzymes. This substitution allows precise nucleotide conversion (C to U, A to G) in non-dividing cells through a biochemical reaction that does not require DNA replication, thereby achieving therapeutic applicability in post-mitotic cells while maintaining high editing efficiency.
Data Source
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AI summary
Embodiments herein include engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein (e.g., C2c1) that enhances binding of the of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type. Embodiments disclosed further include viral vectors for delivery of CRISPR-Cas effector proteins. The vectors may be designed to allow packaging of the CRISPR-Cas effector protein within a single vector. Certain embodiments further include delivery vectors, constructs, and methods of delivering larger genes for systemic delivery.