Journal: Nucleic acids research

Article Title: Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

doi: 10.1093/nar/gkae016

Figure Lengend Snippet: Figure 2. CRISPR-Cas12m activity in E. coli cells. (A) Plasmid DNA interference assay in E. coli . To assess transformation efficiency, each E. coli transformant sample was serially diluted (10 ×) and grown on Kn supplemented media at 37 ◦C o v ernight. Effectiv e DNA interference of the targeted plasmid resulted in a reduction in colony forming units (CFU). Data are presented as mean ( n = 3). KanR – kanam y cin resistance, ori – origin of replication, dAsCas12a – AsCas12a R u vC activ e site mutant (D908A). (B) Experimental w orkflo w of the superfolder GFP (sfGFP) fluorescence interference assay in E. coli . (C) sfGFP fluorescence interference experiment in E. coli . To assess transformation efficiency and sfGFP expression, each E. coli transformant sample was serially diluted (10 ×) and grown overnight at 37 ◦C on the arabinose-supplemented media. Effective DNA binding by dAsCas12a and GoCas12m resulted in reduction of sfGFP fluorescence while CFU remained unchanged. NT – non-targeting control. (D) Bacteriophage plaque formation assay in E. coli . To assess the efficiency of plating (EOP), phages were serially diluted (10 ×) and spotted onto lawns of E. coli expressing AsCas12a, dAsCas12a or GoCas12m. Effective defense against phage infection resulted in reduction of plaque-forming units. Data are presented as mean (n = 3). S and A indicate targeted sense and antisense DNA strands, respectively.

Article Snippet: Expression and purification of Cas12 effectors and their RNP complexes For Cas12m and Cas12a protein purification, E. coli DH10B strain was transformed with expression vectors (pMBP-AsCas12a (gift from Jennifer Doudna, Addgene plasmid #113430), pGB060 (produced from pMBP-AsCas12a by introducing D908A modification, using Phusion SiteDirected Mutagenesis Kit (Thermo Fisher Scientific), pTK200, pTK203; Supplementary Table S4 ).

Techniques: CRISPR, Activity Assay, Plasmid Preparation, Transformation Assay, Mutagenesis, Fluorescence, Expressing, Binding Assay, Control, Plaque Formation Assay, Infection

Journal: Nucleic acids research

Article Title: Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.

doi: 10.1093/nar/gkae016

Figure Lengend Snippet: Figure 4. GoABE base editing activity in E. coli and human cells. (A) Schematic representation of the experimental w orkflo w to detect base editing activity in E. coli resulting in chloramphenicol resistance (CmR) gene restoration. (B) Plasmid DNA transf ormation assa y in E. coli . To assess base editing efficiency, each E. coli transformant sample was serially diluted (10 ×) and grown overnight at 37 ◦C on the Cm supplemented media. Recovery of the colonies indicates successful targeted base editing. As a positive control (Ctrl), E. coli were transformed with a plasmid encoding the intact CmR gene. NT and T indicate non-targeting and targeting crRNA constructs, respectively. (C) Sanger sequencing of plasmids obtained from reco v ered E. coli colonies. (D) Experimental w orkflo w of the enhanced GFP (eGFP) reco v ery assa y perf ormed in HEK293T cells. (E) Flo w cytometry results of counting eGFP-positive HEK293T cells after 24, 48 and 72 h. Data are presented as mean ± SD ( n = 3). (F) Base editing at endogenous sites in HEK293T cells. Selected DNA target sites are labeled according to Richter et al. ( 67 ). Gray areas indicate non-A nucleotides at specific positions across all targets. Data are presented as mean ± SD ( n = 3).

Article Snippet: Expression and purification of Cas12 effectors and their RNP complexes For Cas12m and Cas12a protein purification, E. coli DH10B strain was transformed with expression vectors (pMBP-AsCas12a (gift from Jennifer Doudna, Addgene plasmid #113430), pGB060 (produced from pMBP-AsCas12a by introducing D908A modification, using Phusion SiteDirected Mutagenesis Kit (Thermo Fisher Scientific), pTK200, pTK203; Supplementary Table S4 ).

Techniques: Activity Assay, Plasmid Preparation, Positive Control, Transformation Assay, Construct, Sequencing, Cytometry, Labeling