CasY trancRNA Enhances CRISPR DNA Targeting at Low Temperatures
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Solution Overview
Problem
Current CRISPR-Cas systems face challenges in efficiently targeting and cleaving specific DNA sequences, particularly at low temperatures, and there is a need for improved specificity and efficiency in genome editing applications.
Innovation Solution
The use of a CasY transactivating noncoding RNA (trancRNA) and a CasY guide RNA, which interact with the CasY protein to form a ribonucleoprotein complex that provides sequence specificity and enhances targeting efficiency, even at low temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current CRISPR-Cas systems are used for DNA targeting and cleavage, then genome editing capability is achieved, but efficiency and specificity are reduced particularly at low temperatures
Solution Approach 1:
The patent introduces a transactivating noncoding RNA (trancRNA) component that changes the operational parameters of the CRISPR-Cas system. The trancRNA enhances the binding affinity and stability of the guide RNA-CasY complex, allowing efficient DNA cleavage across a broader temperature range including low temperatures, thereby resolving the contradiction between reliability and productivity
2Use of energy by stationary object
If CRISPR-Cas systems operate at low temperatures, then energy consumption is reduced, but targeting efficiency and cleavage effectiveness deteriorate
Solution Approach 1:
The trancRNA acts as an intermediary molecule that facilitates enhanced interaction between the guide RNA and CasY protein. This intermediary component stabilizes the ribonucleoprotein complex at low temperatures without requiring additional energy input, maintaining DNA interference efficiency while preserving the energy-saving benefit of low-temperature operation
3Device complexity
If standard CRISPR-Cas guide RNA is used, then system simplicity is maintained, but sequence specificity and targeting precision are insufficient
Solution Approach 1:
The patent segments the RNA component into two distinct functional elements: the guide RNA that provides sequence specificity and the trancRNA that enhances binding affinity and stability. This segmentation allows each component to be optimized for its specific function while maintaining overall system simplicity and ease of construction
Data Source
AI summary
Provided are compositions and methods that include a CasY transactivating noncoding RNA (trancRNA) (referred to herein as a “CasY trancRNA”), nucleic acids encoding the CasY trancRNA, and/or a modified host cell comprising the CasY trancRNA (and/or a nucleic acid encoding the same). Subject compositions and methods can also include one or more of: (a) a “CasY” protein (also referred to as a CasY polypeptide, a Cas12d protein, and a Cas12d polypeptide), a nucleic acid encoding the CasY protein, and/or a modified host cell comprising the CasY protein (and/or a nucleic acid encoding the same); and (b) a CasY guide RNA (also referred to herein as a “Cas12d guide RNA”) that binds to and provides sequence specificity to the CasY protein, a nucleic acid encoding the CasY guide RNA, and/or a modified host cell comprising the CasY guide RNA (and/or a nucleic acid encoding the same).