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. 2021 May 5;11:9586. doi: 10.1038/s41598-021-89029-2

Figure 2.

Figure 2

In vitro characterization of CaldoCas9 activity, thermostability and PAM specificity. (A) CaldoCas9, a DNA ‘target’ and gRNA containing a spacer complimentary to the target DNA were incubated in various combinations (top, +  = with, – = without) at 55 °C and the products separated on an agarose gel. Scale on left corresponds to the DNA ladder in the first well of the gel. On far right are indicated the expected band sizes of the uncut (7386 bp) and cut (5479 and 1907 bp) DNA. (B) CaldoCas9, a DNA ‘target’ and gRNA containing a spacer complimentary to the target DNA were incubated together at various temperatures (top) and the products separated on an agarose gel. (C) Top-left: three DNA libraries were prepared that contained a ‘target sequence’ that was common to all three libraries (red font) and a partially randomized sequence (3′- sequence, in blue font, randomized bases indicated as N) downstream of the target sequence. Bottom-left: the DNA libraries (carried on a circular plasmid backbone) were separately PCR amplified (primers indicated as P1 and P2) and incubated with CaldoCas9 and gRNA. The products of each were adapter ligated, PCR enriched and sequenced. Right: PAM sequences permissible for CaldoCas9 cleavage, as revealed by the experiment. The agarose gel images (A,B) have been digitally manipulated for clarity. The original images are provided in supplementary file 5.