CRISPR-Cas12a Self-Inactivating Editing via MicroRNA

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

The challenge in genetic editing is the efficient delivery of large CRISPR-Cas proteins alongside crRNA for in vivo editing of multiple cells while minimizing off-target effects and ensuring cell-specific activity.

Innovation Solution

A CRISPR-Cas system with both RNAse and DNase activity, utilizing a Cas12a protein and a targeting sequence with a direct repeat and guide nucleotide sequence, combined with a microRNA target site for cell-specific activity, delivered via viral vectors or RNA-based replicons to achieve precise genome editing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If CRISPR-Cas proteins are delivered alongside crRNA for in vivo editing, then genome editing efficiency is improved, but delivery complexity and off-target effects increase

Engineering Contradiction:

Improvegenome editing efficiencyVSAvoiddelivery complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent combines the CRISPR-Cas protein and crRNA into a single delivery unit using viral vectors or RNA-based replicons. This merging approach simplifies the delivery process by eliminating the need for separate delivery of protein and RNA components, thereby reducing delivery complexity while maintaining editing efficiency

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent uses viral vectors or RNA-based replicons as intermediary carriers to deliver the CRISPR-Cas system components into target cells. These intermediaries facilitate efficient intracellular delivery of both the Cas protein and crRNA, solving the delivery complexity problem while enabling effective genome editing

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If CRISPR-Cas expression is maintained for longer periods, then editing coverage is improved, but off-target effects and chromosomal translocations increase

Engineering Contradiction:

Improveediting coverageVSAvoidoff-target effects

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent implements temporary, controlled expression of CRISPR-Cas systems rather than continuous long-term expression. By using viral vectors or replicons that provide transient expression, the system achieves sufficient editing coverage during the active period while automatically limiting the duration to prevent accumulation of off-target effects and chromosomal translocations

Inventive Principle:

Principle #19Periodic action

Solution Approach 2:

The patent performs genome editing in a controlled, time-limited manner before potential harmful effects can accumulate. By delivering the system temporarily and allowing completion of editing events within a safe window, the approach achieves necessary editing coverage while preemptively avoiding the harmful consequences of prolonged expression

Inventive Principle:

Principle #10Preliminary action

3Productivity

If CRISPR-Cas systems are delivered for broad cell editing, then editing coverage is improved, but cell-specificity and control are reduced

Engineering Contradiction:

Improveediting coverageVSAvoidcell-specificity

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The patent incorporates cell-specificity mechanisms into the CRISPR-Cas delivery system, such as using promoters or targeting signals that are active only in specific cell types. This allows the system to achieve broad coverage within the desired cell population while maintaining specificity and avoiding off-target effects in other cell types

Inventive Principle:

Principle #3Local quality

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 enables efficient, cell-specific, and precise genome editing with reduced off-target effects, allowing for the modification of target sequences in eukaryotic cells, including human cells, while ensuring the system self-inactivates after function, preventing genomic integration.

Implementation Method 1

at least one microRNA target site capable of hybridizing with a microRNA that mediates cleavage of the microRNA-target site

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

microRNA that mediates cleavage of the microRNA-target site

Methodology Applied

Scientific EffectCleavage:

Implementation Method 3

Cas12a and a subset of other also have RNase function. The RNase function is responsible for processing the pre-crRNA by cleaving direct repeat sequences

Methodology Applied

Scientific EffectRNase activity: Enzyme

Implementation Method 4

Cas12a and a subset of other also have RNase function... the crRNA that is generated as a result of these processing events is sufficient for instilling specificity onto the DNase activity of Cas12a

Methodology Applied

Scientific EffectDNase activity: Enzyme

Implementation Method 5

a guide nucleotide sequence encoding or comprising a crRNA sequence capable of hybridizing with a target sequence and forming a complex with the CRISPR-Cas protein

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS20230088902A1Cell specific, self-inactivating genomic editing using crispr-CAS systems having rnase and dnase activity

Publication Date: 2023.03.23 MT SINAI SCHOOL OF MEDICINE

AI summary

This disclosure provides a CRISPR-Cas system with both RNase and Dnase activity for genetic editing and methods of use thereof. The disclosed CRISPR-Cas system can function in a cell-specific manner, which enables in vivo editing while mitigating the risk of off-target effects.