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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY.

David A Armstrong^1,2,3, Taylor R Hudson¹, Christine A Hodge^2,3

¹Research Service, V.A. Medical Center, White River Junction, VT, USA, 05001.

Biorxiv : the Preprint Server for Biology

|January 30, 2023

Summary

This summary is machine-generated.

Researchers optimized the PlmCas12e gene editing system for targeting the CCR5 gene. This advancement aids in developing therapies for HIV-1 resistance by understanding CasX2 cleavage requirements.

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Substrate Generation for Endonucleases of CRISPR/Cas Systems

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

Area of Science:

Molecular Biology
Gene Editing
Microbiology

Background:

CRISPR/Cas systems are evolving as therapeutic gene editing tools.
Alternative CRISPR systems, including compact Cas12e, offer diverse gene editing capabilities.
The CCR5 gene is a key target for HIV-1 resistance gene therapies.

Approach:

Investigated PlmCas12e (CasX2) endonuclease activity.
Optimized guide RNA lengths and protospacer adjacent motif (PAM) sequences for CCR5 gene cleavage.
Analyzed PlmCas12e cleavage variations based on target site, guide RNA length, and PAM sequence.

Key Points:

PlmCas12e exhibits a PAM preference for purines (A, G) over pyrimidines (T, C) at the fourth position of the TTCN PAM.
Cleavage efficiency is influenced by target site, guide RNA length, and PAM terminal nucleotide.
Understanding CasX2 cleavage requirements is crucial for developing CCR5-targeted gene therapies.

Conclusions:

Optimized PlmCas12e conditions enhance its utility for gene editing applications.
This research advances the development of therapies aimed at conferring HIV-1 resistance via CCR5 gene modification.
The findings contribute to the broader field of CRISPR-based gene therapeutics.