Journal: Nature Communications

Article Title: Genome-wide specificity of dCpf1 cytidine base editors

doi: 10.1038/s41467-020-17889-9

Figure Lengend Snippet: Plasmids encoding versions of dLbCpf1-BE containing mutations in the APOBEC1 domain (YE1: W90Y + R126E; YE2: W90Y + R132E; EE: R126E + R132E; YEE: W90Y + R126E + R132E) were transfected into HEK293T cells to determine if these mutations would improve base editing specificity. Base editing frequencies at on- and -off target sites were determined by targeted deep sequencing. The specificity ratios were calculated by dividing (base editing frequency of dLbCpf1-BE variants at on-target/that at off-target) by (base editing frequency of dLbCpf1-BE-WT at on-target/that at off-target). Mismatched bases and PAM sequences are shown in red and blue, respectively. An RNA bulge is represented as a dash and a DNA bulge is shown in green. Data are shown as mean ± s.e.m. from three biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: The plasmid encoding dLbCpf1-BE for bacterial expression (pET-dLbCpf1-BE, Addgene, #154256) and plasmids encoding dLbCpf1-BE with APOBEC1 variants for mammalian expression (pCMV-dLbCpf1-BE-YE1, Addgene, #154145; pCMV-dLbCpf1-BE-YE2, Addgene, #154146; pCMV-dLbCpf1-BE-EE, Addgene, #154147; and pCMV-dLbCpf1-BE-YEE, Addgene, #154148) are available from Addgene.

Techniques: Transfection, Sequencing

Journal: Nature Communications

Article Title: Genome-wide specificity of dCpf1 cytidine base editors

doi: 10.1038/s41467-020-17889-9

Figure Lengend Snippet: a , b Plasmids encoding matched and mismatched crRNAs (containing 1- to 4-nt mismatches relative to the on-target site) targeting sites in CDKN2A ( a ) and DYRK1A ( b ) were transfected into HEK293T cells together with plasmids encoding dLbCpf1-BE or LbCpf1. Base editing and indel frequencies were measured using targeted deep sequencing. Mismatched bases and PAM sequences are shown in red and blue, respectively. The relative activity (the editing frequency with the mismatched crRNA divided by that with the matched crRNA) indicates the mismatch tolerance. Mismatched crRNAs for which the relative activity of dLbCpf1-BE is at least three times higher than that of LbCpf1 are indicated with blue asterisks; those for which the relative activity of LbCpf1 is at least three times higher are indicated with red asterisks. Data are shown as mean ± s.e.m. from three biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: pET-dLbCpf1-BE, a plasmid encoding a human codon-optimized dLbCpf1-BE with a His purification tag at the N terminus (His 6 -NLS-APOBEC1-XTEN-dLbCpf1(D832A + E925A + D1148A)-NLS-UGI-NLS), was generated using NEBuilder ® HiFi DNA Assembly Master Mix (New England Biolabs) to insert dLbCpf1-BE from pCMV-dLbCpf1-BE (Addgene, #107685) into the pET28a vector (Novagen). pCMV-dLbCpf1-BE (Addgene, #107685) was modified to incorporate mutations in the LbCpf1 domain (N256A, N260A, S348A, K514A, K881A, or K897A; these mutations respectively correspond to N278A, N282A, S376A, K523A, K949A, or K965A in AsCpf1) and combinations of mutations in the APOBEC1 domain (YE1: W90Y + R126E; YE2: W90Y + R132E; EE: R126E + R132E; YEE: W90Y + R126E + R132E) using site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs). crRNA-encoding plasmids were constructed by ligation (Quick Ligation Kit, New England Biolabs) of annealed oligonucleotides to pU6-Lb-crRNA (Addgene, #78957) digested with BsmBI.

Techniques: Transfection, Sequencing, Activity Assay