Fig. 3 | Nature Communications

Fig. 3

From: A thermostable Cas9 with increased lifetime in human plasma

Fig. 3

Small RNA-seq and sgRNA engineering for GeoCas9. a Small RNA sequenced from G. stearothermophilus mapped to the CRISPR locus. Inset shows enlargement of the region corresponding to the tracrRNA and the most highly transcribed repeat and spacer sequence. b Distribution of the length of the spacer sequences extracted from the small RNA sequencing results. c Length optimization of the tracrRNA and crRNA for GeoCas9 and the optimal guide RNA design (right). The length of the tracrRNA, crRNA:tracrRNA duplex and spacer was optimized sequentially by transcribing variations of the sgRNA and testing their ability to guide GeoCas9-mediated cleavage of a radiolabeled substrate. For spacer length, two different targets were used (Target 1 and Target 2). The mean k cleave ± S.D. is shown and experiments were conducted in triplicate

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