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Updated: Sep 15, 2025

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

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ENHANCED CLEAVAGE OF GENOMIC CCR5 USING CASX2^Max.

Christine A Hodge^1,2, Niles P Donegan³, David A Armstrong^1,2

¹Department of Dermatology, Dartmouth Health, Lebanon, NH, USA.

Biorxiv : the Preprint Server for Biology

|July 17, 2025

Summary

This summary is machine-generated.

The enhanced CasX2Max gene editing system shows superior cleavage efficiency compared to native CasX2, offering a promising tool for therapeutic applications like HIV-1 treatment.

Keywords:

CCR5 CRISPR/Cas CasX2 guide RNA

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Published on: March 31, 2022

Area of Science:

Molecular Biology
Gene Editing Technologies
Biochemistry

Background:

CRISPR/Cas systems are powerful tools for gene editing.
CasX2 (PlmCas12e) is a novel CRISPR system with potential therapeutic advantages.
Existing Cas9 systems (SpCas9, SaCas9) have limitations.

Purpose of the Study:

To compare the gene-editing efficiency of native CasX2 and a variant, CasX2Max.
To evaluate their effectiveness in cleaving the CCR5 gene.
To assess their potential for therapeutic applications.

Main Methods:

Utilized CasX2 and CasX2Max systems with varying single-guide RNA (sgRNA) lengths.
Targeted the CCR5 gene, crucial for HIV-1 infection.
Employed Nanopore sequencing for cleavage analysis.
Performed structural modeling to understand activity differences.

Main Results:

Native CasX2 was ineffective at cleaving genomic CCR5.
CasX2Max demonstrated robust CCR5 cleavage with 20 nt and 23 nt sgRNAs.
Structural modeling revealed enhanced sgRNA-DNA stability and catalytic site alignment in CasX2Max.
CasX2Max consistently outperformed native CasX2 in all assays.

Conclusions:

CasX2Max is a significantly more active and efficient gene-editing platform than native CasX2.
The observed improvements are attributed to specific amino acid substitutions enhancing molecular interactions.
CasX2Max represents a superior tool for gene editing therapies, including CCR5 modification for HIV-1 resistance.