Cas9 Heterodimer Small Molecule Dimerizer Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for modifying chromosomal sequences using RNA-guided endonucleases are limited in their ability to efficiently target specific sequences and control gene editing processes, particularly in applications requiring precise regulation of enzyme activity.
Innovation Solution
A Cas9 heterodimer system comprising two fusion polypeptides, a nuclease lobe and an alpha-helical lobe, which can be induced to dimerize by a small molecule dimerizer, allowing for targeted binding and modification of nucleic acids without the need for stem loops 2 and 3 in the guide RNA, enabling precise control over gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a single polypeptide Cas9 is used, then the enzyme has full nuclease activity, but it is difficult to control gene editing precision and regulation
Solution Approach 1:
The Cas9 polypeptide is divided into two separate polypeptides: a first polypeptide containing the nuclease lobe (with RuvC and HNH domains) and a second polypeptide containing the alpha-helical lobe (with PAM-interacting domain). This segmentation allows independent control of nuclease activity and target recognition, improving gene editing precision while enabling regulatory control through dimerization.
2Adaptability or versatility
If the Cas9 heterodimer is designed with separate nuclease and recognition domains, then control over gene editing is improved, but the device complexity increases
Solution Approach 1:
A dimerization domain is introduced as an intermediary component that mediates the interaction between the first polypeptide (nuclease lobe) and the second polypeptide (alpha-helical lobe). This intermediary enables controlled dimerization through small molecule inducers or protein-protein interaction domains, providing versatile control over gene editing while managing system complexity through modular design.
3Measurement precision
If traditional guide RNA with stem loops 2 and 3 is used, then the RNA-guided endonuclease functions properly, but the system lacks precision in controlling enzyme activity
Solution Approach 1:
The guide RNA structure is simplified by removing stem loops 2 and 3, extracting only the essential target-binding functionality. This reduction in guide RNA complexity is compensated by the heterodimeric Cas9 structure, which provides the additional regulatory control and precision needed for accurate enzyme activity management.
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
The Cas9 heterodimer system enables efficient and precise modification of target nucleic acids, including cleavage and transcriptional modulation, with reduced nuclease activity and enhanced specificity, facilitating advanced genetic engineering applications.
Implementation Method 1
Putyrski & Schultz (FEBS Letters 586 (2012) 2097-2105 reviews the use of rapamycin e.g. as a chemical inducer of dimerization, stemming from its ability to bind to two proteins/protein domains simultaneously.
Data Source
Figure 1A
Figure 1B
Figure 2
AI summary
The present disclosure provides a Cas9 heterodimer, as well as nucleic acids encoding the Cas9 heterodimer, and host cells comprising the nucleic acids. The present disclosure provides a system that includes a Cas9 heterodimer of the present disclosure and at least one of: a Cas9 guide RNA, and a dimerizing agent.. A Cas9 heterodimer of the present disclosure is useful in a wide variety of applications, which are also provided.