Fig. 1 | Nature Communications

Fig. 1

From: Engineering of CRISPR-Cas12b for human genome editing

Fig. 1

Identification of mesophilic Cas12b nucleases. a Locus schematics and protein domain structure highlighting the differences between Cas9, Cas12a, and Cas12b nucleases. Crystal structures of SpCas9 (PDB:4oo8 [10.2210/pdb4OO8/pdb]), AsCas12a (PDB:5b43 [10.2210/pdb4OO8/pdb]), and AacCas12b (PDB:5u30 [10.2210/pdb5U30/pdb]). b In vitro reconstitution of Cas12b systems with purified Cas12b protein and synthesized crRNA and tracrRNA identified through RNA-Seq. Reactions were carried out at the indicated temperatures for 90 min and 250 nM Cas12b protein. c, d AkCas12b and BhCas12b indel activity in 293T cells with six sgRNA variants. Error bars represent s.d. from n = 4 replicates. See Supplementary Fig. 3b, c for sgRNA sequences. e Schematic of BhCas12b sgRNA design 1 with gray shading highlighting the location of changes to the guide design (see Supplementary Fig. 3 for exact sequences of guide design variants 2–6). Source data are provided as a Source Data file

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