Cell culture and gene modification analysis
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 in Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 10% fetal bovine serum. sgRNA plasmid (500 ng) and effector [nuclease, BE3, or ABE(7.10)] plasmid (500 ng) were transfected into cells as duplicates (2 × 105 per well in a 24-well plate) with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco). After 48 hours posttransfection, genomic DNA was extracted using QuickExtract Solution (Epicentre), and genomic loci were amplified by PCR using the KAPA HiFi HotStart ReadyMix (Kapa Biosystems). For base-editing analysis, amplicons were purified and submitted for Sanger sequencing (Genewiz). For indel analysis, the T7E1 reaction was conducted according to the manufacturer’s instructions, and equal volumes of products were analyzed on a 2% agarose gel stained with SYBR Safe (Thermo Fisher Scientific). Unprocessed gel image files were analyzed in Fiji (41). The cleaved bands of interest were isolated using the rectangle tool, and the areas under the corresponding peaks were measured and calculated as the fraction cleaved of the total product. Percent gene modification was calculated as follows (42)Embedded Image

All samples were performed in duplicates, and percent gene modifications were averaged. SD was used to calculate error bars.

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