u6 promoter  (New England Biolabs)

Journal: Nature Communications

Article Title: Circular RNA-mediated inverse prime editing in human cells

doi: 10.1038/s41467-025-59120-7

Figure Lengend Snippet: a Schematic diagram of circular RNA-mediated inverse prime editors (ciPEs). b Schematic diagrams of the structure of ciPE editors. CMV, the CMV promoter of cytomegalovirus; U6, the polymerase III promoter of U6; NLS, bipartite nuclear localization signal; M-MLV RTΔRNase H, deletion variant of M-MLV RT with no RNase H domain; 5′ RL, 5′ ribozyme and ligation sequences; 3′ RL, 3′ ribozyme and ligation sequences; RTT, reverse transcriptase template; PBS, primer binding site. c Comparison of inverse prime editing frequencies between split iPE2 and ciPE2 at four target sites in HEK293T cells. Frequencies (mean ± s.e.m.) in c were obtained from three biological replicates ( n = 3). d Comparison of inverse prime editing efficiencies between ciPE2–5 and split iPEmax2–5 using epegRNA at six target sites in HEK293T cells. Frequencies (mean ± s.e.m.) in d were obtained from four biological replicates ( n = 4). circRNA, circular RNA; Ins, insertion; Del, deletion. InDels, byproducts of random insertions and deletions. P values were obtained from two-tailed Student’s t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The sgRNA, nicking sgRNA, pegRNA, and epegRNA vectors driven by the human U6 promoter were constructed by annealing oligonucleotides and inserting them into the pOsU3 backbone (Addgene #170132) using the Golden Gate assembly method.

Techniques: Variant Assay, Ligation, Reverse Transcription, Binding Assay, Comparison, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Mechanism of Cas9 inhibition by AcrIIA11

doi: 10.1093/nar/gkaf318

Figure Lengend Snippet: AcrIIA11 inhibits SaCas9 in vitro and in human HEK293T cells. ( A ) Agarose gel and ( B ) quantification of DNA cleavage by SaCas9. Graphs represent the mean of three replicates. Error bars: S.E.M. ( C ) HEK293Ts are transiently transfected with plasmids carrying SaCas9 + sgRNA. A second plasmid encodes either AcrIIA4 (positive control) or AcrIIA11. ( D ) Representative agarose gel and ( E ) quantification of indel percentage for the CACNA1D site. Error bars are the standard deviation of three replicates. P -values (not significant [ns], P > 0.05; *** P < 0.001; **** P < 0.0001) were determined using a Student’s t -test. ( F ) Quantification of the editing efficiency when AcrIIA11 or AcrIIA4 are present relative to when no Acr is expressed. Error bars are the standard deviation of three replicates. P -values (not significant [ns], P > 0.05; **** P < 0.0001) were determined using a Student’s t -test.

Article Snippet: The Sa Cas9 and CMV promoter-driven AcrIIA4 expression vectors were purchased from Addgene (Plasmid #85452 and #113038) [ , ].

Techniques: In Vitro, Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Positive Control, Standard Deviation