Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene.

Arildo Nerys-Junior,Luciene P Braga-Dias,Paula Pezzuto,Vinícius Cotta-De-Almeida,Amilcar Tanuri

doi:10.1590/1678-4685-gmb-2017-0065

Abstract

The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells.

Highlights

HIV-1 entry into target CD4+ cells requires the C-C chemokine receptor type 5 (CCR5) that acts as a co-receptor for the V3 loop of the gp120 viral adhesion protein (Hill et al, 1997)
The resulting 445 bp amplicon containing transcription activator-like effector nucleases (TALEN) and CRISPRCas9 sites was cloned into a pGEM®-T Easy Vector (Promega) and transformed in E. coli JM109 (Promega) competent cells that were plated on an ampicillin/XGal/isopropyl b-D-1-thiogalactopyranoside (IPTG) plate
A portion of cells was separated before cell sorting for subsequent extraction of genomic DNA and the amplicon containing TALEN and CRISPR-Cas9 target cloned into a pGEM®-T Easy Vector for subsequent comparison between sorted and unsorted cells

Summary

Introduction

HIV-1 entry into target CD4+ cells requires the C-C chemokine receptor type 5 (CCR5) that acts as a co-receptor for the V3 loop of the gp120 viral adhesion protein (Hill et al, 1997). A CCR5 natural 32-bp deletion (defined as the CCR5D32 allele) is an effective restriction condition against HIV-1 infection. As this mutant allele produces a truncated protein that is not expressed on the cell surface, individuals homozygous for CCR5D32. Antiretroviral therapy was discontinued, resulting in a rapid decrease in viral load followed by long-term viral absence. This was considered the first functional cure for HIV-1 infection, with the patient remaining functionally healed

Methods

Results

Conclusion