Type VII CRISPR Effector Targeting for Specific Genome Editing
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Solution Overview
Problem
There is a need for novel genome engineering technologies that are affordable, easy to set up, scalable, and capable of targeting multiple positions within the eukaryotic genome, as existing methods like designer zinc fingers and TALEs are limited in versatility and accessibility.
Innovation Solution
Development of Type VII CRISPR effector proteins with a RuvC-like nuclease domain and a crRNA comprising a direct repeat sequence, designed to hybridize with target nucleic acids, forming a CRISPR complex that can modify nucleic acid sequences, including cleavage, base substitution, or modulate gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing genome-editing techniques (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbations can be produced, but the technology is limited in versatility and accessibility, and remains complex and difficult to set up
Solution Approach 1:
The CRISPR-Cas system uses a single programmable effector protein (Cas9) that can be directed to different genomic locations by changing the guide RNA sequence, making it universally applicable to multiple genome positions without requiring different protein tools for each target
Solution Approach 2:
The guide RNA acts as an intermediary between the user-specified target sequence and the Cas9 effector protein, translating biological sequence information into physical targeting guidance, thereby simplifying the system setup and improving accessibility
2Ease of manufacture
If existing genome-editing techniques are used, then targeted genome perturbations can be produced, but the technology is difficult to set up and scale
Solution Approach 1:
The system is segmented into separate functional components: a reusable Cas9 protein and disposable guide RNAs. This allows the complex protein to be produced once and reused, while the simple guide RNAs can be easily scaled and customized for different targets, improving both ease of setup and scalability
Solution Approach 2:
The system achieves versatility and scalability by changing the sequence parameter of the guide RNA rather than changing the protein structure, allowing rapid reconfiguration for different genomic targets without re-producing the complex effector protein
3Measurement precision
If CRISPR-Cas systems are used, then nucleic acid targeting is achieved, but off-target effects may occur
Solution Approach 1:
The system incorporates PAM sequence recognition as a feedback verification step, where the Cas9 protein checks for the presence of a specific protospacer adjacent motif (PAM) sequence adjacent to the target site before activating cleavage, thereby ensuring specificity and reducing off-target 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 Type VII CRISPR system enables precise and targeted modification of nucleic acid sequences, including cleavage and gene expression modulation, with improved specificity and reduced off-target effects, suitable for various cell types including human, plant, and yeast cells.
Implementation Method 1
a crRNA comprising a direct repeat sequence and a guide sequence that is designed to hybridize to a target nucleic acid sequence
Implementation Method 2
Effectors containing a RuvC-like nuclease domain and an HNH nuclease are categorized as Type II
Data Source
AI summary
The present application provides systems, methods and compositions used for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of Type VII Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems and transposable elements.