Type V-A CRISPR Compositions for Efficient Transgene Integration
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Solution Overview
Problem
There is a need for improved CRISPR-Cas systems that provide high integration and expression efficiency of transgenes with high post-transfection cell viability in eukaryotic cells.
Innovation Solution
The development of CRISPR-Cas based compositions and methods that incorporate protecting groups, donor template-recruiting sequences, and editing enhancers into Type V-A CRISPR systems, along with the use of polynucleotides with covalently closed ends and nuclear localization signals to enhance nuclear import and editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional CRISPR-Cas systems are used for genome editing, then genome targeting capability is achieved, but transgene integration efficiency and expression efficiency are insufficient
Solution Approach 1:
The patent segments the CRISPR-Cas system into distinct functional components: a CRISPR-Cas nuclease complex for genome targeting, a separate donor template for transgene integration, and specific polynucleotide sequences with protecting groups and recruiting elements. This segmentation allows each component to be optimized independently for its specific function, thereby improving both integration efficiency and expression reliability.
Solution Approach 2:
The patent introduces intermediary elements including polynucleotides with covalently closed ends that serve as vectors for delivering the CRISPR-Cas system and donor templates into cells. These intermediaries facilitate efficient transgene integration by mediating between the genome targeting function and the transgene expression function, resolving the contradiction between integration efficiency and expression reliability.
2Productivity
If CRISPR-Cas systems are used for genome editing, then editing capability is achieved, but post-transfection cell viability is reduced
Solution Approach 1:
The patent modifies key parameters of the CRISPR-Cas system by using engineered nucleases with altered PAM recognition specificities and by optimizing the sequence and structure of donor templates. These parameter changes enable the system to achieve high editing capability while minimizing harmful effects on cell viability following transfection.
Solution Approach 2:
The patent incorporates protecting groups and recruiting sequences into the polynucleotide design before transfection. These protective elements cushion against potential harmful effects during the editing process, thereby maintaining high cell viability while still achieving effective genome editing.
3Ease of operation
If standard polynucleotides are used for delivery, then delivery capability is achieved, but nuclear import efficiency is insufficient
Solution Approach 1:
The patent performs preliminary action by incorporating nuclear localization signals (NLS) into the polynucleotide structure before delivery into the cell. This preliminary modification ensures that once the polynucleotide enters the cell, it is efficiently directed to the nucleus, thereby resolving the contradiction between delivery capability and nuclear import efficiency.
4Productivity
If conventional CRISPR systems are used, then basic genome targeting is achieved, but editing precision and efficiency are limited
Solution Approach 1:
The patent applies local quality by introducing editing enhancers and specific polynucleotide sequences at the target site location. These localized modifications enhance the precision and efficiency of genome editing at the specific target location, while the rest of the system remains optimized for general delivery and expression, thereby resolving the contradiction between editing efficiency and targeting precision.
Data Source
AI summary
CRISPR-Cas systems have been engineered for various purposes, such as genomic DNA cleavage, base editing, epigenome editing, and genomic imaging. Although significant developments have been made, there still remains a need for new and useful CRISPR-Cas systems as powerful precise genome targeting tools. The invention disclosed herein comprises CRISPR-Cas based compositions and methods for high integration efficiency and expression efficiency of transgenes together with high post-transfection cell viability in eukaryotic cells.