Class 2 CRISPR-Cas Systems with PAM Recognition Domains
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Solution Overview
Problem
Current CRISPR-Cas systems face limitations in specificity and efficiency for targeted nucleic acid cleavage, particularly in recognizing and cleaving non-target DNA sequences, which can lead to off-target effects and reduced efficacy in genome editing applications.
Innovation Solution
The development of Class 2 CRISPR-Cas systems, including Cas12c and CasY proteins, which form ribonucleoprotein complexes with guide RNAs to specifically target and cleave DNA sequences, utilizing transactivating noncoding RNAs (trancRNAs) to enhance precision and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas systems are used for targeted nucleic acid cleavage, then genome editing capability is achieved, but off-target effects occur due to reduced specificity
Solution Approach 1:
The patent introduces a PAM recognition domain that provides localized specificity at the target site. The effector protein is engineered to recognize specific PAM sequences (e.g., 5'-TTTN-3') adjacent to the target sequence, creating a dual-specificity system where both the guide RNA and PAM domain must match their respective targets. This local quality enhancement ensures that cleavage only occurs at precise locations, eliminating off-target effects while maintaining genome editing capability.
Solution Approach 2:
The patent creates composite effector proteins by fusing the CRISPR-Cas nuclease domain with a PAM recognition domain. This composite structure integrates two functional elements: the guide RNA-binding capability and the PAM sequence recognition. The fusion protein acts as a unified molecular complex that simultaneously requires both guide RNA complementarity and PAM sequence match for activation, thereby achieving high specificity while preventing off-target cleavage events.
2Productivity
If CRISPR-Cas systems target DNA sequences, then genome editing is achieved, but cleavage efficiency is reduced
Solution Approach 1:
The patent implements a preliminary recognition step where the effector protein first binds to the PAM sequence before proceeding to cleave the target DNA. This preliminary action serves as a verification checkpoint: the protein must successfully recognize and bind the PAM sequence (5'-TTTN-3' or variants) before the nuclease domain is activated. This two-step process ensures high precision by confirming target identity before cleavage, while the efficient PAM binding mechanism maintains overall productivity by enabling rapid target verification.
3Object-affected harmful factors
If effector proteins are designed for high specificity, then off-target effects are reduced, but system complexity increases
Solution Approach 1:
The patent designs the PAM recognition domain to recognize a family of related sequences (5'-TTTN-3' where N can be any nucleotide, plus variants like 5'-TTTA-3', 5'-TTTC-3', 5'-TTTG-3', 5'-TTTG-3'). This universal recognition capability allows a single effector protein design to handle multiple PAM variants, reducing the need for separate engineered proteins for each sequence variation. The multi-functionality of the PAM domain maintains high specificity across different target sites without proportionally increasing system complexity.
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 systems demonstrate improved specificity and efficiency in cleaving target DNA sequences, reducing off-target effects and enhancing the precision of genome editing, while also being adaptable for use in various cellular environments.
Implementation Method 1
a CRISPR/Cas guide RNA that binds to and provides sequence specificity to the effector protein
Implementation Method 2
Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein (the effector protein) bound to RNA is responsible for binding to and cleavage of a targeted sequence
Data Source
AI summary
Provided are compositions and methods that include one or more of: (1) a Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the effector protein, and/or a modified host cell comprising the effector protein (and/or a nucleic acid encoding the same); (2) a CRISPR/Cas guide RNA that binds to and provides sequence specificity to the Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the CRISPR/Cas guide RNA, and/or a modified host cell comprising the CRISPR/Cas guide RNA (and/or a nucleic acid encoding the same); and (3) a CRISPR/Cas transactivating noncoding RNA (trancRNA), a nucleic acid encoding the CRISPR/Cas trancRNA, and/or a modified host cell comprising the CRISPR/Cas trancRNA (and/or a nucleic acid encoding the same).