PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding

Abstract

Cas12a is a class 2 type V CRISPR-associated nuclease that uses an effector complex comprised of a single protein activated by a CRISPR-encoded small RNA to cleave double-stranded DNA at specific sites. Cas12a processes unique features as compared to other CRISPR effector nucleases such as Cas9, and has been demonstrated as an effective tool for manipulating complex genomes. Prior studies have indicated that DNA flexibility at the region adjacent to the protospacer-adjacent-motif (PAM) contributes to Cas12a target recognition. Here, we adapted a SELEX-seq approach to further examine the connection between PAM-adjacent DNA flexibility and off-target binding by Cas12a. A DNA library containing DNA-DNA mismatches at PAM + 1 to + 6 positions was generated and subjected to binding in vitro with FnCas12a in the absence of pairing between the RNA guide and DNA target. The bound and unbound populations were sequenced to determine the propensity for off-target binding for each of the individual sequences. Analyzing the position and nucleotide dependency of the DNA-DNA mismatches showed that PAM-dependent Cas12a off-target binding requires unpairing of the protospacer at PAM + 1 and increases with unpairing at PAM + 2 and + 3. This revealed that PAM-adjacent DNA flexibility can tune Cas12a off-target binding. The work adds support to the notion that physical properties of the DNA modulate Cas12a target discrimination, and has implications on Cas12a-based applications.

Fig. 1

A schematic of SELEX-seq adapted for analyzing Cas12a binding to a library of DNA duplexes containing DNA-DNA mismatch(es). The duplexed DNA library contains a FnCas12a PAM and randomized DNA-DNA mismatch(es) at the PAM + 1 to + 6 positions. “N” indicates the randomized nucleotides at the non-target strand and “*” indicates a 5’ fluorescence tag (5’-FAM) at the target strand. The library was subjected to binding to a Cas12a effector with an RNA guide (red line) that does not complement the DNA protospacer (blue lines) (Step 1). The bound and unbound DNA populations were separated using a native gel shift assay (Step 2). The individual DNAs in the corresponding populations were identified by sequencing (Step 3). The sequencing data were analyzed to obtain the propensity of binding for each individual DNA (Step 4).

Fig. 2

Example of a SELEX-seq dataset. (A) An example of separation of the bound and unbound DNA. As shown binding was carried out with 100 nM duplexed DNA library and 50 nM dCas12a effector complex (formed with a protein/RNA ratio of 1:2) (see Methods). Note that to account for the weak off-target binding, the effector concentration was set much higher than the reported 12 nM on-target K_d of FnCas12a. The bound and unbound DNAs were separated on a native polyacrylamide gel with a 5% and a 10% segment and visualized with fluorescence imaging. “*” indicated an imaging artifact at intersections of the gel segments. (B) Enrichment analysis of the bound (, Eq. 2) and unbound (, Eq. 3) populations recovered from the gel shown in panel (A). (C) Relative enrichment values, (Eq. 4), calculated for data shown in panel (B).

Fig. 3

Correlation between off-target binding and the total number of mismatches. The relative enrichment for groups of sequences containing a given total number of mismatches (r(#MM)) was plotted against the total number of mismatches. The data shows off-target binding increases with the total number of PAM-adjacent mismatches. See additional information in SI Sect. S3.2.1.

Fig. 4

Positional preference analysis of the 5MM group of sequences. (A) for different positional mismatch arrangements, with PAM identified by a yellow box, mismatched nucleotides by a red box, and matched nucleotides by a black box. Numbers 1–6 represent the position of the randomized nucleotides. (B) Examples of individual 5MM sequences with positive and therefore favor off-target binding. (C) Examples of individual 5MM sequences that form a dT/dG₁ wobble pair. (D) Examples of individual 5MM sequences with misfolded PAM. For panels (B), (C), and (D), the individual sequence number, nominal DNA-DNA mismatch pattern, , and values are indicated. The predicted most stable secondary structures are shown, with magenta filled nucleotides indicating PAM, white filled nucleotides indicate the 6 randomized nucleotides, green circled nucleotides indicate mismatched nucleotides, and the PAM + 1 to + 6 positions are marked. See additional information in SI Sect. S3.2.2.

Fig. 5

Analysis of nucleotide preferences. (A) for all possible nucleotides at the randomized positions for different numbers of mismatches represented as a heatmap. (B) Web logo plot of position-dependent nucleotide preferences obtained via multiple linear regression (MLR) analysis. Further details are described in SI. Sect. S3.4.