CRISPR-Cas System Targeting Conserved Genomic Sequences
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Solution Overview
Problem
Current technologies lack an effective method for selectively killing bacterial and archaeal cells based on specific genomic sequences, particularly those with conserved target sequences adjacent to a protospacer adjacent motif (PAM).
Innovation Solution
The introduction of a recombinant nucleic acid construct comprising a CRISPR array with repeat sequences and spacer nucleotides that are complementary to target sequences in bacterial and archaeal cells, along with a Type I-E CRISPR associated complex for antiviral defense (Cascade complex) and Cas3 polypeptide, to specifically target and kill cells with conserved genomic sequences adjacent to a PAM.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used for bacterial cell killing, then broad-spectrum activity is achieved, but specificity to target cells with conserved genomic sequences is lost
Solution Approach 1:
The CRISPR array is designed with specific spacer sequences that are complementary to conserved genomic regions adjacent to PAM sites in target bacteria. This localized sequence matching enables the system to distinguish target cells from non-target cells, achieving high specificity while maintaining the ability to target multiple bacterial species that share these conserved sequences
2Manufacturing precision
If non-specific killing methods are used, then ease of operation is maintained, but precision in targeting specific bacterial sequences is compromised
Solution Approach 1:
The CRISPR array is pre-designed with spacer sequences that are complementary to the target bacterial genome sequences adjacent to PAM sites. This preliminary configuration of the guide RNA ensures that upon introduction into the cell, the system immediately achieves precise targeting without requiring additional operational steps for sequence selection or optimization
3Reliability
If CRISPR-Cas system is introduced, then sequence-specific killing capability is achieved, but system complexity increases
Solution Approach 1:
The Type I-E CRISPR-Cas system is introduced as a self-contained ribonucleoprotein complex that includes all necessary components (Cascade complex, Cas3 endonuclease, and crRNA) to perform sequence-specific DNA targeting and cleavage. The system autonomously recognizes target sequences adjacent to PAM sites and executes killing without requiring external cellular machinery or additional complex regulatory elements
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
This approach enables precise killing of bacterial and archaeal cells with specific genomic targets, ensuring high specificity and efficiency by leveraging the CRISPR-Cas system's sequence-specific targeting capabilities.
Implementation Method 1
each of the one or more spacer sequences is complementary to a target sequence (protospacer) in the genome of the bacterial and/or archaeal cells of the population
Implementation Method 2
a Type I-E CRISPR associated complex for antiviral defense (Cascade complex) and Cas3 polypeptide, to specifically target and kill cells with conserved genomic sequences adjacent to a PAM
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 killing one or more cells in a population of bacterial and/or archaeal cells.