Cas13 RNA-Targeting System for Transcript Knockdown
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Solution Overview
Problem
Current applications of CRISPR-Cas nucleases in Drosophila melanogaster are limited to DNA-targeting class 2 systems, and there is a need for improved methods to target RNA for transcriptome engineering and gene knockdown applications.
Innovation Solution
The development of nucleic acid molecules encoding a Cas13 polypeptide and a guide RNA with spacers and Cas13-specific direct repeats, capable of specifically hybridizing with target RNAs, along with methods for modifying target RNAs in cells using these molecules, enabling programmable RNA-targeting systems like CasRx for transcript knockdown and genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-Cas nucleases are used for DNA-targeting applications, then genome editing capability is achieved, but RNA-targeting capability is lost
Solution Approach 1:
The patent divides the CRISPR system into separate functional components: a DNA-targeting Cas nuclease (e.g., Cas9) for genome editing and a separate RNA-targeting guide RNA (gRNA) system for transcript knockdown. This segmentation allows each component to be optimized for its specific function while working together in an integrated platform, resolving the contradiction between DNA and RNA targeting capabilities.
Solution Approach 2:
The patent creates a universal CRISPR-based platform that can perform multiple functions: DNA targeting for genome editing using Cas nucleases and RNA targeting for transcript knockdown using guide RNAs. This multi-functional system addresses the need for both DNA and RNA targeting capabilities within a single integrated approach, improving adaptability without compromising DNA-targeting reliability.
2Measurement precision
If traditional RNA interference methods are used for gene knockdown, then RNA targeting is achieved, but specificity and off-target effects are worsened
Solution Approach 1:
The patent incorporates feedback mechanisms through the use of guide RNAs with carefully designed spacers that are processed and folded into hairpin structures. The Cas13 protein binds to these guide RNAs and uses them to specifically recognize and cleave target RNAs through complementary base pairing. This feedback loop ensures high specificity by requiring precise spacer-target RNA complementarity, thereby reducing off-target effects while maintaining high targeting precision.
Solution Approach 2:
The patent changes the key parameter of spacer length and sequence composition in the guide RNA to optimize targeting precision. By adjusting spacer length (typically 20-40 nucleotides) and sequence characteristics, the system achieves high specificity for target RNAs while minimizing off-target activity. This parameter optimization allows the system to distinguish between true targets and similar sequences, improving measurement precision.
3Ease of operation
If Cas13 polypeptide and guide RNA are expressed separately, then protein expression control is improved, but system complexity increases
Solution Approach 1:
The patent merges the Cas13 polypeptide coding sequence and guide RNA sequence into a single nucleic acid molecule, where the guide RNA is encoded within or adjacent to the Cas13 open reading frame. This merging simplifies the delivery and expression system by reducing the number of separate genetic elements that need to be managed, while still allowing independent control of protein expression through promoter selection and regulatory elements. The combined molecule reduces device complexity while maintaining ease of operation.
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
This approach allows for efficient on-target transcript knockdown with limited off-target activity, demonstrating clear phenotypes and providing a platform for RNA-targeting applications in vivo, paving the way for alternative gene knockdown studies and potential antiviral strategies.
Implementation Method 1
the spacers are capable of specifically hybridizing with one or more target RNAs
Implementation Method 2
Cas13 polypeptide... enabling programmable RNA-targeting systems like CasRx for transcript knockdown
Data Source
AI summary
Provided herein are compositions and methods for CRISPR based RNA-targeting. The compositions include nucleic acid molecules comprising a sequence encoding a Cas13 polypeptide and a sequence encoding a guide RNA comprising one or more spacers and one or more Cas13-specific direct repeats, where the spacers are capable of specifically hybridizing with one or more target RNAs. The disclosure further provides methods of modifying a target RNA in a cell and transgenic organisms.