Microbial CRISPR-Cas Endonucleases With Diverse PAM Specificity
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Solution Overview
Problem
There is a need for a larger variety of Cas endonucleases for target-specific modification of polynucleotides in both prokaryotic and eukaryotic cells, as existing CRISPR-Cas systems may be limited in their range and specificity.
Innovation Solution
The development of CRISPR-Cas systems derived from various microbial strains, including Pseudarthrobacter chlorophenolicus, Microbacterium sp., and Bacillus megaterium, which can create double-strand breaks in target polynucleotides and are directed by guide polynucleotides for precise editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing CRISPR-Cas systems are used for genome editing, then target-specific modification capability is achieved, but the variety and range of available Cas endonucleases are limited
Solution Approach 1:
The patent discovers and characterizes multiple novel Cas endonucleases (Cas12C, Cas12D, Cas12E, Cas12F, Cas12G, Cas12H) from different bacterial species that all perform the same genome editing function but with different PAM recognition specificities. This multi-functionality approach expands the toolbox of available Cas endonucleases while maintaining the core genome editing capability, directly addressing the need for greater variety and range in CRISPR-Cas systems.
Solution Approach 2:
The patent systematically varies key parameters of Cas endonucleases including PAM recognition sequences (5'-TTTN-3' for Cas12C, 5'-TTTV-3' for Cas12D, 5'-TTCN-3' for Cas12E, etc.), target sequence specificity, and cleavage patterns. By changing these parameters across different bacterial sources, the patent creates a diverse family of Cas endonucleases that can target different genomic locations and sequences, thereby expanding the overall range and adaptability of CRISPR-Cas systems.
2Measurement precision
If Cas endonucleases with different PAM preferences are developed, then target specificity is improved, but the complexity of selecting and characterizing new endonucleases increases
Solution Approach 1:
The patent segments the CRISPR-Cas system into distinct functional components: guide RNA molecules that provide target recognition and Cas endonucleases that perform cleavage. By separating these functions and independently optimizing each component's PAM preferences and target specificities, the patent reduces the complexity of selecting complete systems while maintaining high target specificity through modular assembly of guide RNA-Cas enzyme pairs.
Solution Approach 2:
The patent introduces guide RNA molecules as intermediaries between the Cas endonuclease and the target DNA sequence. The guide RNA mediates specific binding to target sequences adjacent to different PAM motifs, allowing the Cas endonuclease to achieve high target specificity without requiring direct complex interaction with diverse PAM sequences. This intermediary approach simplifies the selection process by decoupling PAM recognition from the catalytic core.
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
These microbial-derived CRISPR-Cas systems enable precise and targeted modifications in plant, animal, and fungal cells, enhancing genome editing capabilities.
Implementation Method 1
a target polynucleotide that comprises a sequence that is complementary to a guide polynucleotide
Implementation Method 2
capable of creating a double strand break in, or adjacent to, a target polynucleotide
Data Source
AI summary
The disclosure relates to novel CRISPR-Cas systems identified in a variety of bacterial species. The compositions identified herein may be used to edit a heterologous polynucleotide, for example in a eukaryotic or prokaryotic cell.