[ADRB2 Gene Knockout in Human Primary T Cells by Multiple sgRNAs Construced using CRISPR/Cas9 Technology]

Area of Science:

• Molecular Biology
• Immunology
• Gene Editing

Background:

• The beta-2 adrenergic receptor (β2-AR) plays a role in T cell function.
• Efficiently knocking out the ADRB2 gene in primary human T cells is crucial for studying its function.

Purpose of the Study:

• To establish a rapid and efficient method for knocking out the ADRB2 gene in human primary T cells using CRISPR/Cas9 technology.
• To investigate the efficacy of a multiple single-guide RNA (sgRNA) strategy for gene editing.

Main Methods:

• Designed and cloned six paired sgRNAs targeting the ADRB2 gene into expression vectors.
• Tested sgRNA cleavage efficiency in HEK-293T cells using T7EN I digestion.
• Constructed a recombinant plasmid expressing four efficient sgRNAs targeting different ADRB2 sites.
• Transfected human primary T cells with the multiple sgRNAs and Cas9 plasmids via electroporation.
• Assessed gene knockout efficiency using flow cytometry (FCM).

Main Results:

• The multiple sgRNA strategy yielded higher gene editing efficiency and diverse mutation types (insertions, deletions, large fragment rearrangements) compared to single sgRNAs.
• Achieved 100% mutation efficiency in targeted T cells.
• FCM analysis showed a reduction in β2-AR positive cells from 43.09% in control cells to 25.61% in cells treated with the multiple sgRNA strategy.

Conclusions:

• A simple and operable method for knocking out the ADRB2 gene in human primary T cells was established.
• This gene editing approach provides a valuable tool for further investigating the role of β2-AR in human T cells.