Type I-E CRISPR Array Screening for Resistant Variant Cells
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Solution Overview
Problem
Existing methods for screening variant cells are limited in their ability to efficiently identify and select cells that are resistant to specific target sequences while avoiding unintended cleavage, particularly in the context of bacterial and other organisms.
Innovation Solution
A recombinant CRISPR-Cas system is introduced to create a population of cells, which includes a recombinant nucleic acid construct comprising a CRISPR array and a Type I-E CRISPR system, which comprises a population of cells, which includes a recombinant nucleic acid construct comprising a CRISPR array comprising a recombinant nucleic acid construct encoding a Type I-E CRISPR associated complex and a Cas3 polypeptide, allowing for the selection of variant cells resistant to a selection marker.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing screening methods are used to identify variant cells, then the process is simpler, but the ability to efficiently identify and select cells resistant to specific target sequences while avoiding unintended cleavage is limited
Solution Approach 1:
The CRISPR-Cas system is divided into distinct functional modules: a Type I-E Cascade complex (comprising multiple Cas proteins: Cas5, Cas6, Cas7, Cas8, Cas9) and a separate CRISPR array encoding guide RNAs. This segmentation allows independent optimization of target recognition (Cascade complex) and guide RNA generation (CRISPR array), improving screening precision while managing complexity through modular design
Solution Approach 2:
The patent introduces a Type I-E Cascade complex as an intermediary between the CRISPR array (guide RNA source) and the target DNA sequences. This intermediary complex processes the guide RNAs and uses them to specifically recognize and bind target sequences, enabling precise identification of variant cells without direct contact between the CRISPR array and target DNA, thereby avoiding unintended cleavage
2Reliability
If a recombinant CRISPR-Cas system is introduced to improve cell selection capability, then the ability to select variant cells resistant to specific target sequences is improved, but the system complexity increases
Solution Approach 1:
The Type I-E CRISPR-Cas system is designed with multi-functionality: the Cascade complex can process multiple different CRISPR arrays with various spacer sequences, enabling a single system architecture to screen for multiple different target sequences across different experiments. This universality improves selection reliability for identifying resistant variant cells while avoiding the need to design entirely new systems for each target, thereby managing complexity
Solution Approach 2:
The system performs preliminary action by introducing the complete Type I-E CRISPR-Cas system (including Cascade complex and CRISPR array) into the cell population before the actual screening process. The Cascade complex is pre-assembled and activated, and the CRISPR array is pre-transcribed to generate guide RNAs, so that when target sequences are present, the system is immediately ready to identify and select resistant variant cells, improving reliability while the one-time introduction manages overall complexity
3Object-affected harmful factors
If existing methods are used for cell screening, then the process is faster and simpler, but the ability to avoid unintended cleavage of non-target sequences is reduced
Solution Approach 1:
The CRISPR array is designed with high local quality through the use of spacer sequences that are specifically complementary to target sequences, with each spacer engineered to match only its intended target. This local sequence specificity, combined with the Type I-E Cascade complex's requirement for both guide RNA complementarity and PAM sequence recognition, ensures that cleavage occurs only at the precise target location, avoiding unintended cleavage of non-target sequences while the modular design manages system complexity
Data Source
AI summary
This invention relates to recombinant Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and recombinant nucleic acid constructs encoding Type I-E CASCADE complexes, plasmids, retroviruses and bacteriophage comprising the same, and methods of use thereof for screening for variant cells of an organism.