Extended Data Fig. 5: Experimental assessment of insertion tolerance in Cas12a. | Nature Methods

Extended Data Fig. 5: Experimental assessment of insertion tolerance in Cas12a.

From: Rational engineering of allosteric protein switches by in silico prediction of domain insertion sites

Extended Data Fig. 5

a, Insertion scores are mapped onto a cryo-electron microscopy (cryo-EM) structure of MbCas12a. PDB ID: 6IV6. b, c, HEK293T cells were transfected with vectors encoding (i) the indicated Cas12a-PDZ insertion variant, (ii) a firefly luciferase targeting gRNA and (iii) a luciferase reporter. Samples were incubated for 48 hours, and luciferase activity was measured in a plate reader. The activity of insertion variants predicted to be active (b) or inactive (c) is shown. Bars indicate means, error bars the standard deviation, and black dots individual data points from n = 3 independent experiments. nt, non-targeting gRNA.

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