Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity

Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2′,4′-BNANC[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNANC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA–DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, “zipped” conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.

Effect of BNA NC -modified crRNAs on the ability of nuclease-deficient Cas9 (dCas9) to bind DNA target sequences.

Supplementary Figure 11
Effect of BNA NC modifications on crRNA/DNA melting temperature.

Supplementary Figure 12
Kinetic analysis of time spent in open to zipped transition and docked states using single-molecule FRET.

Supplementary Figure 13
Cas9 in vitro cleavage kinetics using either WAS-RNA or WAS-BNA-3 crRNAs on several target sequences.

Supplementary Figure 14
Cas9 in vitro cleavage kinetics using either WAS-RNA or WAS-BNA-2 crRNAs on several target sequences.

Supplementary Figure 16
Incorporation of LNA into crRNAs improves Cas9 cleavage specificity in vitro.

Supplementary Figure 17
Distribution of mutations in pre-and post-selection libraries following in vitro library selection with unmodified or LNA-modified crRNAs.

Supplementary Figure 18
In vitro specificity profiling results for LNA-modified crRNAs.

Supplementary Figure 19
Comparison of indel size resulting from Cas9 DNA cleavage with unmodified or LNA-modified crRNAs.

Supplementary Figure 20
Distribution of indel location using unmodified or LNAmodified crRNAs on WAS target.

Supplementary Figure 21
Distribution of indel location using unmodified or LNAmodified crRNAs on EMX1 target.

Supplementary Figure 22
Effect of LNA-modified crRNAs on activity and kinetics of Cas9 in vitro.

Supplementary Figure 23
Model of Cas9 structure highlighting interactions between crRNA nucleotides 10-14 and Cas9. Supplementary Table 1 In vitro cleavage assay data used to generate Fig. 1. Supplementary Table 2 In vitro cleavage assay data used to generate Supplementary Fig. 1. Supplementary Table 3 In vitro cleavage assay data used to generate Supplementary Fig. 2. Supplementary Table 4 Statistics of sequences from in vitro high-throughput library selection experiment. Supplementary Table 5 Statistics of cellular modification frequencies, sample size and P-values for high-throughput sequencing of Cas9:gRNA cleavage in U2OS-Cas9 and HeLa-Cas9 cells using no guide RNA, unmodified, BNA NC -or LNAmodified crRNAs. Supplementary Table 6 In vitro cleavage assay data used to generate Supplementary Fig. 16. Supplementary Table 7 Cellular modification rates induced by unmodified or LNA-modified crRNAs targeting WAS or EMX1. Supplementary Table 8 Unmodified, BNA NC -and LNA-modified crRNA and tracrRNA sequences used. Supplementary Table 9 Oligonucleotides used in this study.  -OT1  WAS-OT2  WAS-OT3  WAS-OT4  WAS-OT5   target   EMX1  EMX1-OT1  EMX1-OT2  EMX1-OT3  EMX1-OT4  EMX1- lettering. Targets which were highly cleaved in vitro are indicated in red, while targets No. of mutations   Reactions in which no cleavage products were observed are marked as undetected (UD).  .

T G G A T G G A G G A A T G A G G A G T N G G
nM Cas9 RNP complex. A score of zero indicates no change in specificity. Difference in specificity was calculated as, specificity score BNA NC -specificity score RNA .     Values showing in vitro cleavage specificity for unmodified crRNA and 9 BNA NC -modified crRNAs towards either WAS or EMX1 on-and off-target sequences (as listed Fig. 1b, c);

Supplementary
Mean ± SE (n = 2). Experiments were performed using 150 nM Cas9 RNP and 5 nM DNA.  Values showing in vitro cleavage specificity for unmodified crRNA and 9 BNA NC -modified crRNAs towards either WAS or EMX1 on-and off-target sequences (as listed Fig. 1b, c);

Supplementary
Mean ± SE (n = 2). Experiments were performed using 15 nM Cas9 RNP and 5 nM DNA. Values showing in vitro cleavage specificity for unmodified crRNA and 9 BNA NC -modified crRNAs towards either WAS (left) or EMX1 (right) on-and off-target sequences containing single-nucleotide mismatches (as listed in Supplementary Figure 2); Mean ± SD (n = 2).

Supplementary
Experiments were performed using 15 nM Cas9 RNP and 5 nM DNA. A plus sign (+) indicates that the following nucleotide is a BNA NC , while an asterisks (*) indicates the following nucleotide is a LNA. Nucleotides with a preceding (r) were ordered as RNA.

The following sequences were used for single-molecule FRET experiments
WAS smFRET /5Biosg/ttt ttt GAG GAA G/iAmMC6T/G CCT TGG ATG GAG GAA TGA GGA GTT GGC TCC CAT CAC ATC WAS-OT4 smFRET /5Biosg/ttt ttt GAG GAA G/iAmMC6T/G CCT AGG AGG GAG GAA TGG GGA GTT GGC TCC CAT CAC ATC Sequence names ending with a number sign (#) were ordered from IDT as dsDNA gBlocks. An asterisks (*) indicates that the preceding nucleotide was incorporated as a hand mix of bases consisting of 79 mol % of the intended base, and 7 mol % of each of the other three bases.