Supplementary Figure 4: Biochemical measurements of crRNA affinity and dual catalytic activity of LbaCas13a.
From: Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme
a, Filter-binding assays measuring the affinity of LbaCas13a for its cognate crRNA. The quantified binding data was fit to a standard binding isotherm with measured dissociation constants (mean ± st. dev., n= 3) of 0.98 ± 0.08 nM for a cognate crRNA and 84.7 ± 12.6 nM for a representative off target ssRNA. b-c, The dependence of LbaCas13a ssRNA targeting activation on spacer sequence length (20-28 nt) was measured by fluorescent cleavage assays. Reactions were carried out as described in the Methods, except that cleavage buffer was used instead of processing buffer. (b) Apparent rates (mean ± st. dev., n= 3) were fitted to resulting time courses and (c) endpoint fluorescent values (background corrected mean ± st. dev., n= 3) were measured. All crRNA with various spacer lengths were able to direct LbaCas13a for ssRNA cleavage, although the shortest spacer tested (20-nt) reached a lower plateau value while retaining a similar apparent rate compared to all other spacer lengths. d, pre-crRNA processing assays were performed as described in the Methods for alanine substitutions of residues interacting with the 5′ end of the mature crRNA in the LbaCas13a binary structure. Quantified data were fit to single-exponential decays with calculated pseudo-first order rate constants (kobs) (mean ± st. dev., n= 3) as follows: Lba Wildtype (WT), 0.109 ± 0.005 min−1; Lba W325A, 0.011 ± 0.004 min−1; and Lba N1232A, 0.014 ± 0.005 min−1. Rates could not be calculated for H328A, K432A, K435A, K1305A, and K1320A. Endpoint data from these curves is presented in Fig 4. e-f, ssRNA targeting by LbaCas13a and alanine substitution mutations was carried out consistent with b-c. Asterisks mark mutants for which rates could not be fit. Binding affinity of LbaCas13a mutants to (g) pre-crRNA dG(-29) (pre-crRNA mimic) or (h) mature crRNA substrates measured by filter-binding. Measured binding affinities were fit to a standard binding isotherm and are summarized in (i). j, Representative gel of pre-crRNA processing by LbaCas13a in the presence and absence of divalent metal ion chelator, EDTA for 60 mins. k, pre-crRNA processing by LbaCas13a carried out as described in the Methods for a pre-crRNA substrate bearing 8- or 2-nt 5′ to the scissile phosphate.
