Type I CRISPR-Cas Editing for Long-Range Chromosomal Deletions
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Solution Overview
Problem
Existing CRISPR-Cas systems are inefficient for long-range chromosomal deletions and deletions screens in eukaryotic cells, particularly at physiological temperatures.
Innovation Solution
Utilization of Type I CRISPR-Cas systems from Thermobifida fusca, comprising a multi-subunit ribonucleoprotein complex (Cascade) and helicase-nuclease enzyme Cas3, to introduce long-range deletions in human embryonic stem cells and HAP1 cells, with guide RNA targeting and efficient DNA modification at temperatures up to 37°C.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Type I CRISPR-Cas systems are used for genome editing in eukaryotic cells, then long-range chromosomal deletions can be introduced with high editing efficiency, but the system complexity increases due to the multi-subunit ribonucleoprotein complex Cascade and helicase-nuclease enzyme Cas3
Solution Approach 1:
The Type I CRISPR-Cas system is divided into distinct functional modules: the multi-subunit Cascade complex for target recognition and binding, and the separate Cas3 helicase-nuclease for DNA degradation. This segmentation allows each component to be optimized independently while working together to achieve long-range deletions with high efficiency in eukaryotic cells
Solution Approach 2:
The Cascade complex serves multiple functions: it binds guide RNA, recognizes target DNA sequences, facilitates R-loop formation, and recruits Cas3 to the target site. This multi-functionality consolidates what would otherwise require separate proteins, managing system complexity while maintaining high productivity for long-range genome manipulations
2Adaptability or versatility
If Type I CRISPR-Cas systems are used to create long-range deletions, then a spectrum of deletions from hundreds to 100 kilobases can be generated, but the precision of deletion boundaries becomes more challenging to control
Solution Approach 1:
The system dynamically adjusts deletion outcomes based on multiple factors including guide RNA sequence, target locus context, and cellular conditions. The processive nature of Cas3 allows the deletion spectrum to adapt to different genomic locations and sequences, enabling versatile long-range deletions while accepting variable boundary precision that can be optimized through guide RNA design
Solution Approach 2:
By changing parameters such as guide RNA sequence composition, Cas3 mutations (e.g., N23A in Cse2), and delivery methods, the system can modulate deletion characteristics. The N23A mutation in Cse2 specifically enhances function at physiological temperatures while maintaining the ability to generate a spectrum of deletions from hundreds to 100 kilobases
3Productivity
If CRISPR systems are optimized for high editing efficiency, then 5%-95% editing efficiency can be achieved, but off-target effects and harmful factors increase
Solution Approach 1:
The system replaces the mechanical cutting action of Cas9 with a biochemical process involving Cascade-mediated R-loop formation followed by Cas3 helicase-nuclease activity. This substitution allows for more controlled DNA degradation starting from a defined position, achieving high editing efficiency while reducing off-target effects through the stepwise mechanism of target recognition before degradation
4Ease of operation
If conventional CRISPR systems are used at physiological temperatures, then cell compatibility is improved, but editing efficiency decreases
Solution Approach 1:
The system employs parameter changes through specific protein mutations, particularly the N23A mutation in the Cse2 subunit of Cascade, which alters thermal sensitivity. This mutation enables the system to function efficiently at physiological temperatures (37°C) while maintaining high editing efficiency, resolving the contradiction between cell compatibility and productivity
Solution Approach 2:
The system uses a composite approach combining thermally stable Cas3 enzyme with the modified Cascade complex. This composite system leverages the temperature resilience of Cas3 while the N23A-mutated Cascade provides optimized binding at physiological temperatures, achieving both cell compatibility and high editing efficiency simultaneously
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
Achieves editing efficiencies of 5%-95% with deletions ranging from a few hundred base-pairs to 100 kilobases, preserving the target site and demonstrating improved editing efficiency and specificity compared to previous systems.
Implementation Method 1
a guide RNA (a targeting RNA) comprising a sequence that is complementary to a targeted site in a segment of the DNA
Implementation Method 2
the helicase-nuclease enzyme Cas3 to degrade DNA processively
Implementation Method 3
the helicase-nuclease enzyme Cas3 to degrade DNA processively
Implementation Method 4
Cascade to identify DNA targets, and the helicase-nuclease enzyme Cas3 to degrade DNA processively
Implementation Method 5
modifying the DNA by nicking, causing a double stranded break (DSB), and/or unidirectional deleting of a single strand of the DNA
Data Source
AI summary
Provided are compositions, methods, and kits for CRISPR-based editing of DNA targets by Type I CRISPR-associated (Cas) enzymes.