CRISPR Base Editing CCR5 Gene HIV Protection
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Solution Overview
Problem
Current methods for creating CCR5 loss-of-function mutants in vivo are ineffective due to the large number of cells that need to be modified, and are associated with off-target effects, genome instability, and oncogenic modifications.
Innovation Solution
The use of CRISPR/Cas9-based base-editing technology to precisely target and modify the CCR5 or CCR2 genomic locus, resulting in loss-of-function variants with a low probability of off-target effects and minimal impact on genomic stability, by contacting the encoding polynucleotide with a fusion protein comprising a guide nucleotide sequence-programmable DNA binding protein domain and a cytosine deaminase domain.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing methods are used to create CCR5 loss-of-function mutants in vivo, then the CCR5 gene can be modified, but the large number of cells that need to be modified makes the method ineffective
Solution Approach 1:
The invention changes the mechanism of genetic modification from traditional nuclease-based methods to base editing, which directly converts C to T or G to A at specific positions without requiring double-strand breaks. This parameter change in the editing mechanism enables effective CCR5 mutation with fewer cell modifications needed
2Reliability
If traditional genome-editing treatments are used, then CCR5 can be modified, but off-target effects, genome instability, or oncogenic modifications may be caused
Solution Approach 1:
The invention extracts and removes the harmful nuclease domain from the CRISPR system, using only the guide RNA and Cas9 nickase or dCas9 for targeted base modification. This eliminates the double-strand breaks and associated off-target effects while retaining the ability to precisely edit the CCR5 gene
Solution Approach 2:
The invention introduces base editors as intermediary molecules that directly convert bases without creating double-strand breaks. These base editors serve as mediators between the guide RNA and the target DNA, enabling precise CCR5 mutation while avoiding the harmful effects of traditional nuclease-based editing
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 approach generates CCR5 and CCR2 loss-of-function variants that provide protection against HIV infection, reduce symptoms, and delay the progression of HIV to AIDS, while minimizing safety concerns such as oncogene activation or tumor suppressor inactivation.
Implementation Method 1
contacting results in deamination of the target C base by the fusion protein, resulting in a cytosine-guanine pair (C:G) to thymine-adenine pair (T:A) change in the CCR5-encoding polynucleotide
Data Source
AI summary
Provided herein are systems, compositions, and methods of introducing protective and/or loss-of-function variants of CCR5 and CCR2. Variants may be introduced using a CRISPR/Cas9-based nucleobase editor or other guide nucleotide sequence-programmable DNA binding protein domain-based fusion protein described herein. Further provided herein are compositions and methods of preventing and treating conditions related to HIV infection and progression as well as to AIDS.