ascas12a protein  (Addgene inc)

Journal: ACS Omega

Article Title: CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity

doi: 10.1021/acsomega.9b01469

Figure Lengend Snippet: Reconstituting the Cas12a enzyme. (A) Schematic of a Cas12a/RNA/DNA construct, with the RNA-a guide sequence shown in blue and the red wedges marking the DNA cleavage sites (see Supporting Information Sections S1–S3 for more details). (B) Schematics of DNA duplex substrates (see also Table S3 ), with “R/D” indicating complementarity between the RNA guide and the t-strand of the DNA protospacer, and “D/D” indicating pairing between DNA t- and nt-strand. (C,D) Representative gels of cleavage of duplexes i–iv (10 nM each) by effector complex (100 nM) of LbCas12a/LbRNA-a or AsCas12a/AsRNA-a, respectively. Reactions were monitored using 5′- 32 P labeled t-strand, with precursors marked by “■” and t-strand products marked by “▶”.

Article Snippet: AsCas12a protein was obtained commercially from Integrated DNA Technologies (Coralville, Iowa, #1081068).

Techniques: Construct, Sequencing, Labeling

Journal: ACS Omega

Article Title: CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity

doi: 10.1021/acsomega.9b01469

Figure Lengend Snippet: Assessing DNA binding to Cas12a. (A) Schematic of the competition assay. (B) Schematics of the competitor duplexes (see also Table S3 ). (C,D) Representative gels of competitions with LbCas12a/LbRNA-a and AsCas12a/AsRNA-a, respectively. Data shown were obtained with 1 nM probe i* ( 32 P-labeled t-strand), 10 nM enzyme, and 1 μM competitors.

Article Snippet: AsCas12a protein was obtained commercially from Integrated DNA Technologies (Coralville, Iowa, #1081068).

Techniques: Binding Assay, Competitive Binding Assay, Labeling

Journal: ACS Omega

Article Title: CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity

doi: 10.1021/acsomega.9b01469

Figure Lengend Snippet: Assessing binding to Cas12a by DNA with PAM-adjacent bubbles. Shown in (A) is a schematic of the competition assay. Shown in (B) are schematics of the competitor duplexes (see also Table S3 ). Shown in (C,D) are representative gels of competitions with LbCas12a/LbRNA-a and AsCas12a/AsRNA-a, respectively. Data shown were obtained with 1 nM probe i* ( 32 P-labeled t-strand), 10 nM enzyme, and 1 μM competitors.

Article Snippet: AsCas12a protein was obtained commercially from Integrated DNA Technologies (Coralville, Iowa, #1081068).

Techniques: Binding Assay, Competitive Binding Assay, Labeling