Base-editing analysis with TLR
This protocol is extracted from research article:
Minimal PAM specificity of a highly similar SpCas9 ortholog
Sci Adv, Oct 24, 2018; DOI: 10.1126/sciadv.aau0766

HEK293T cells were maintained, as previously described, and transfected with the corresponding sgRNA plasmids (333 ng), ABE7.10 plasmids (333 ng), and synthetically constructed TLR plasmids (333 ng) into cells as duplicates (2 × 105 per well in a 24-well plate) with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco). After 5 days posttransfection, cells were harvested and analyzed on a FACSCelesta machine (Becton Dickinson) for mCherry (561-nm laser excitation, 610/20 filter for detection) and GFP (488-nm laser excitation, 530/30 filter for detection) fluorescence. Cells expressing mCherry were gated, and percent GFP+ calculation of the subset were calculated. All samples were performed in duplicates, and percentage values were averaged. SD was used to calculate error bars. The TLR spacer sequence is 5′-TTCTGTAGTCGACGGTACCG-3′.

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