Biallelic DNA Modification in Cattle Zygotes

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Solution Overview

Problem

Current methods for biallelic DNA modification in cattle zygotes are inefficient, leading to inconsistent gene knockout phenotypes due to the presence of maternal or nascent mRNA and wildtype alleles.

Innovation Solution

A method involving the use of DNA and RNA modification agents, such as CRISPR-Cas systems, to simultaneously modify target DNA and knockdown target RNA in cattle zygotes, thereby increasing the efficiency of biallelic modifications.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional DNA modification methods are used in cattle zygotes, then DNA modification can be achieved, but biallelic modification efficiency is low and gene knockout phenotypes are inconsistent

Engineering Contradiction:

Improvebiallelic modification efficiencyVSAvoidgene knockout phenotype consistency

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention divides the gene modification process into two independent segments: DNA modification using CRISPR-Cas9 system and RNA knockdown using CRISPR-Cas13 system. This segmentation allows simultaneous targeting of both alleles through DNA cleavage while independently suppressing maternal mRNA through RNA interference, thereby achieving consistent biallelic modification efficiency

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention merges two modification systems (CRISPR-Cas9 for DNA and CRISPR-Cas13 for RNA) into a single integrated approach delivered together to cattle zygotes. This combination enables simultaneous action on both DNA and RNA levels, overcoming the limitation of conventional single-system approaches that fail to achieve reliable biallelic knockouts due to persistent maternal mRNA

Inventive Principle:

Principle #5Merging (Combining)

2Productivity

If maternal or nascent mRNA is present in zygotes, then normal development can proceed, but biallelic DNA modification efficiency is reduced

Engineering Contradiction:

Improvebiallelic modification efficiencyVSAvoidmaternal or nascent mRNA interference

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The invention converts the harmful effect of maternal mRNA persistence into a beneficial outcome by using CRISPR-Cas13 to specifically target and knockdown maternal mRNA. The presence of maternal mRNA, which normally interferes with modification efficiency, is transformed into a target for RNA interference, thereby eliminating the harmful factor and improving biallelic modification efficiency

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Solution Approach 2:

The invention performs preliminary RNA knockdown action before completing DNA modification. By delivering CRISPR-Cas13 system simultaneously with CRISPR-Cas9, the maternal mRNA is degraded in advance, creating a favorable environment for subsequent DNA cleavage and biallelic modification, thus overcoming the interference of maternal mRNA

Inventive Principle:

Principle #10Preliminary action

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 proposed method significantly enhances the efficiency of biallelic polynucleotide modifications in cattle zygotes, overcoming the limitations of existing technologies by effectively managing maternal or nascent mRNA and achieving consistent genomic modifications.

Implementation Method 1

modifying a target DNA using a DNA modification agent; the DNA modification agent is a CRISPR-Cas system, a TALEN, a Zinc Finger Nuclease

Methodology Applied

Scientific EffectNuclease activity: Enzyme

Implementation Method 2

knocking down target RNA using an RNA modification agent; the RNA modification agent is a CRISPR-Cas system or RNAi

Methodology Applied

Scientific EffectRNA interference:

Implementation Method 3

the RNA modification agent is a CRISPR-Cas13 system, a CRISPR-Cas13a system

Methodology Applied

Scientific EffectRNA degradation: Enzyme

Implementation Method 4

delivery of the DNA modifying agent and/or RNA modifying agent occurs via electroporation

Methodology Applied

Scientific EffectElectroporation:

Data Source

PatentUS20250084442A1Methods of biallelic modification

Publication Date: 2025.03.13 VIRGINIA TECH INTELLECTUAL PROPERTIES INC

AI summary

Described herein are methods and techniques for biallelic modification of DNA.