Engineered Cas13 Proteins for Programmable RNA Editing in Eukaryotes
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Solution Overview
Problem
Current gene editing systems, such as CRISPR/Cas9, are limited to targeting DNA and cannot efficiently edit RNA in vivo, necessitating the development of a system capable of targeted RNA editing.
Innovation Solution
Identification and engineering of novel Cas13 proteins, particularly Cas13g3, which are optimized for RNA cleavage and programmable RNA editing, combined with CRISPR/Cas13 systems to form a gene editing system that can target and modify RNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Weight of stationary object
If type VI-D CRISPR/Cas13 system is used for RNA editing, then the system has smaller protein size and better prospects in AAV-based gene therapy, but the system requires PFS sequence for targeted editing which is not present in eukaryotes
Solution Approach 1:
The patent modifies the Cas13 protein by introducing point mutations (e.g., D389A, D392A in Cas13d) to alter its catalytic activity and PFS requirement. These parameter changes enable the protein to function in eukaryotic systems without the restrictive PFS sequence requirement while maintaining RNA cleavage capability
Solution Approach 2:
The patent separates the Cas13 protein into different variants (Cas13d, Cas13g, Cas13h, Cas13i, Cas13j, Cas13k) with different properties, and further creates engineered versions (dCas13, ncCas13) by removing specific functional domains. This segmentation allows selection of optimal variants for different applications
2Reliability
If conventional CRISPR/Cas9 system is used, then the system can target DNA genome, but the system cannot perform targeted editing on RNA in vivo
Solution Approach 1:
The patent uses crRNA as an intermediary molecule that bridges the Cas13 protein and target RNA sequence. The crRNA contains a spacer region complementary to the target RNA, enabling specific recognition and binding through base pairing, thus mediating the interaction between the effector protein and the RNA target
3Productivity
If Cas13 proteins are engineered for enhanced RNA cleavage activity, then the editing efficiency is improved, but the system may cause off-target effects
Solution Approach 1:
The patent creates different Cas13 variants (nuclease-active and nuclease-dead) with localized functional differences. The dCas13 variants retain RNA binding capability but lack cleavage activity, allowing them to be used for transcriptional regulation without causing off-target RNA degradation. This local quality differentiation enables precise control of editing effects
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 engineered Cas13 proteins demonstrate high efficiency and specificity in RNA editing, offering potential applications in gene therapy and treatment of diseases associated with nucleic acid mutations.
Implementation Method 1
it uses the principle of complementary nucleic acid base pairing to identify target RNA sequences
Implementation Method 2
guide Cas effector proteins for targeted cleavage
Data Source
AI summary
The present application relates to isolated novel CRISPR/Cas13 proteins, a gene editing systems based thereon, and a method for using said proteins for RNA level gene editing. Provided are non-naturally occurring or engineered RNA targeting systems, and said systems each have a novel Cas13 effector protein that targets RNA and at least one type of guide molecule.