Engineered Cpf1 CRISPR Delivery for Precise Low Off-Target Editing
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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, leading to off-target effects and reduced therapeutic efficacy.
Innovation Solution
Development of engineered CRISPR-Cas effector proteins, particularly Cpf1, with modifications to enhance binding and editing preferences, and the use of optimized delivery vectors to improve specificity, efficacy, and safety by reducing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas systems are used for genome editing, then editing efficacy is improved, but off-target effects increase reducing safety
Solution Approach 1:
The patent modifies parameters of the CRISPR-Cas system including changing the guide RNA sequence composition, adjusting the Cas9 protein structure through amino acid substitutions, and optimizing the PAM sequence requirements. These parameter changes enable the system to maintain high editing efficacy while significantly reducing off-target effects by making the system more discriminating in target recognition
Solution Approach 2:
The patent introduces high mobility group (HMG) domain-containing proteins that locally enhance the specificity of CRISPR-Cas binding at the target site. These proteins create a localized quality improvement in binding specificity without affecting the overall editing efficacy, thereby reducing off-target effects while maintaining on-target activity
2Reliability
If CRISPR-Cas systems are used for genome editing, then therapeutic efficacy is improved, but safety is reduced due to off-target effects
Solution Approach 1:
The patent introduces HMG domain-containing proteins as intermediary molecules that mediate between the CRISPR-Cas complex and the target DNA. These intermediaries enhance the specificity of the interaction, allowing the system to achieve high therapeutic efficacy while minimizing harmful off-target effects by acting as a selective bridge to the intended target
Solution Approach 2:
The patent employs dynamically regulated CRISPR-Cas systems where the activity of the Cas9 protein can be controlled through inducible promoters or small molecule inhibitors. This dynamic control allows the system to maintain high therapeutic efficacy when activated while minimizing off-target effects during the off-state, thereby improving the safety profile
3Ease of operation
If standard CRISPR-Cas systems are used, then ease of operation is maintained, but manufacturing precision of edits is reduced
Solution Approach 1:
The patent segments the CRISPR-Cas system into modular components including separate expression cassettes for the guide RNA, Cas9 protein, and HMG domain proteins. This segmentation allows each component to be independently optimized for precision while maintaining the overall ease of operation through standardized modular assembly and delivery methods
Data Source
AI summary
Embodiments disclosed herein are directed to engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein that enhances binding of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type. In certain example embodiments, the CRISPR-Cas effector protein is a Type V effector protein. In certain other example embodiments, the Type V effector protein is Cpf1. Embodiments disclosed herein are directed to viral vectors for delivery of CRISPR-Cas effector proteins, including Cpf1. In certain example embodiments, the vectors are designed so as to allow packaging of the CRISPR-Cas effector protein within a single vector. There is also an increased interest in the design of compact promoters for packing and thus expressing larger transgenes for targeted delivery and tissue-specificity. Thus, in another aspect certain embodiments disclosed herein are directed to delivery vectors, constructs, and methods of delivering larger genes for systemic delivery.