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. Author manuscript; available in PMC: 2020 Jun 29.
Published in final edited form as: Chembiochem. 2020 Mar 5;21(11):1633–1640. doi: 10.1002/cbic.201900736

Figure 3.

Figure 3.

Crosslinking rationale, scheme and results. A) Crystal structure of Cas9:sgRNA complex (PDB ID: 4UN3).[34] Close up view of RNA–protein interface around G81. Distance between 3′ phosphate to a K31C mutation represented by dashed line. B) Crosslinking reaction scheme between K31C and iodoacetamide (SIA; top) or K31C and maleimide (AMAS; middle) or K31pAzF and cyclooctyne (DBCO; bottom) to form a covalently crosslinked Cas9:sgRNA complex. C) Crosslinking products resolved by SDS PAGE. A gel shift occurs only when the modified sgRNA is in the presence of nucleophilic mutant (K31C, C80S, C574S) of Cas9. (For images of other combinations, see Figure S3.) D) Crosslinking percentage vs. RNA identify plotted. Plotted values correspond to averages±standard deviation (n=3).