Type I CRISPR/Cas System for Precise Eukaryotic Genome Engineering
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Solution Overview
Problem
Current genome editing technologies, particularly in eukaryotic cells, face challenges in precisely regulating genes and achieving high specificity, with Type II CRISPR-Cas systems showing low frequency of large deletions compared to insertions or deletions (indels) and limited exploitation of Type I CRISPR-Cas systems for targeted genome engineering.
Innovation Solution
Development of a Type I CRISPR/Cas system composition comprising a Cascade complex, Cas3 polypeptide, and crRNA, optimized for eukaryotic cells, which includes a nuclear localization signal and codon-optimized polynucleotide sequences, enabling precise genome engineering through targeted nucleic acid constructs and protein-RNA complexes for introducing insertions, deletions, or mutations by homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Type II CRISPR-Cas systems are used for genome editing in eukaryotic cells, then genome editing can be achieved, but the frequency of large deletions is extremely low compared to indels
Solution Approach 1:
The patent changes the fundamental parameters of the CRISPR system by switching from Type II (Cas9) to Type I (Cascade/Cas3) systems, which fundamentally alters the mechanism of genome editing. This parameter change enables both precise editing and high-frequency large deletions simultaneously, resolving the contradiction between precision and productivity.
Solution Approach 2:
The patent employs a composite CRISPR system comprising multiple proteins (Cascade complex and Cas3) working together, rather than a single protein system. This composite approach enables the system to achieve both precise gene targeting and high-frequency large deletions, overcoming the limitations of Type II systems.
2Adaptability or versatility
If Type I CRISPR-Cas systems are used, then diversity and prevalence are high, but little work has been done to exploit them for eukaryotic genome engineering
Solution Approach 1:
The patent segments the Type I CRISPR-Cas system into distinct functional components (Cascade complex and Cas3 protein) that can be independently expressed and optimized for eukaryotic cells. This segmentation enables the system to be adapted to eukaryotic genome engineering while maintaining the diversity and versatility of Type I systems.
Solution Approach 2:
The patent introduces viral delivery systems as intermediaries to facilitate the expression and function of Type I CRISPR-Cas components in eukaryotic cells. This intermediary approach bridges the gap between the natural prokaryotic system and eukaryotic application, making exploitation easier while preserving system diversity.
3Measurement precision
If Cascade complex and Cas3 are used for targeted DNA degradation, then specificity can be achieved, but off-target effects remain a challenge
Solution Approach 1:
The patent incorporates feedback mechanisms through the PAM recognition step, where the Cascade complex continuously verifies target sequence accuracy before activating Cas3. This feedback loop ensures high specificity while minimizing off-target effects, as only perfectly matched targets with correct PAM sequences are processed.
Solution Approach 2:
The patent replaces the simpler Cas9 mechanism with a more complex Cascade/Cas3 system that uses RNA-DNA hybridization and PAM verification to achieve higher specificity. This substitution of the mechanical cutting mechanism with a more verification-based system reduces off-target effects while maintaining productivity.
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 Type I CRISPR/Cas system achieves enhanced specificity and efficiency in genome editing, reducing off-target effects and allowing for precise regulation of gene expression, including the potential for large deletions and chromosomal modifications, thereby overcoming limitations of existing systems.
Implementation Method 1
The spacer portion of the CRISPR RNA then serves as a guide to recognize complementary foreign genetic material
Implementation Method 2
Cas3 then nicks the non-target strand and begins degrading the strand in a 3'-to-5' direction
Implementation Method 3
The CRISPR array is transcribed and processed into individual CRISPR RNAs
Implementation Method 4
The at least one polynucleotide sequence is operably linked to a eukaryotic promoter and comprises a nuclear localization signal
Data Source
AI summary
Disclosed herein are Type I Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system related compositions and methods of using said Type I CRISPR/Cas system related compositions for altering gene expression and genome engineering. The invention relates to compositions comprising Type I CRISPR-Cas polypeptides and CRISPR array nucleic acids designed for genome modification in eukaryotic cells and for targeted killing of eukaryotic cells.