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. 2018 Jul 27;9(1):2962.
doi: 10.1038/s41467-018-05391-2.

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Josh Tycko  1   2 , Luis A Barrera  1   3 , Nicholas C Huston  1   4 , Ari E Friedland  1 , Xuebing Wu  5 , Jonathan S Gootenberg  6 , Omar O Abudayyeh  7 , Vic E Myer  1 , Christopher J Wilson  8 , Patrick D Hsu  9   10
Affiliations

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Josh Tycko et al. Nat Commun. .

Erratum in

Abstract

Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

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Conflict of interest statement

J.T., L.A.B., N.H., A.E.F., J.S.G., V.E.M., C.J.W., and P.D.H. are employees, consultants, or former employees of Editas Medicine. X.W. and O.O.A. declare no competing interests.

Figures

Fig. 1
Fig. 1
Pairwise library screen of SaCas9 genome editing specificity. a Schematic of the pairwise library cassette. Individual library members have variable spacer and target sequences, and each member is identifiable by a unique 15 nt error-correcting Hamming barcode. Individual molecules of each library member are tagged with a unique randomized barcode (rBC). b Schematic of the pooled pairwise library screen workflow. Each guide-target pair is associated with many rBCs. The library was initially installed in HEK 293T cells by lentivirus and sequenced to generate a whitelist of rBCs associated with error-free guide-target pairs. SaCas9 was then delivered by lentivirus. After 3 and 14 days, the library was sequenced to measure Cas9-mediated indels. c Reproducibility of a pairwise library screen increases if a greater number of whitelist rBCs is required for each library member. Heat color represents the number of library members in that hexagonal bin, while white area represents 0 library members. d The fraction of recovered library members decreases as a greater number of rBCs is required. All downstream analyses were performed with a minimum of 20 unique whitelist rBCs for each library member (grey). e On-target indel efficiency on Day 3 for SaCas9 guide-target pairs, binned by spacer length. (n = 653 guide-target pairs). f Comparison of SaCas9 activity across categories of target sites. The scrambled targets are negative controls (n = 47,374 guide-target pairs). c through f use data from Day 3 post-SaCas9 delivery. Boxes in e and f denote median and interquartile range (IQR), and whiskers extend to the 10th and 90th percentile
Fig. 2
Fig. 2
SaCas9 sgRNA with shorter spacers are less tolerant of mismatches. a Average effect of sgRNA spacer length and mismatch position on SaCas9 single-mismatch tolerance. Mean ± 95% confidence interval is shown (n = 16,742 guide-target pairs). b Heatmap of relative SaCas9 cleavage efficiency for each possible RNA:DNA base pair, calculated for all single mismatch library members (n = 10,344 guide-target pairs). c Heatmap of relative SaCas9 double-mismatch tolerance across different spacer lengths and positions. Data is aggregated from 10 sgRNAs for which all possible single and double mismatches were tested (n = 28,707 guide-target pairs). d DNA base identity and position effect on double-mismatch tolerance for 20 nt spacer sgRNA (n = 6017 guide-target pairs) and e for 21 nt spacer sgRNA (n = 6729 guide-target pairs). All panels use data from Day 3 post-SaCas9 delivery
Fig. 3
Fig. 3
SaCas9 specificity model ranks synthetic and endogenous off-target sites. a A regression model trained on the pairwise library screen data correlates with an independent validation set (n = 86 guide-target pairs). The sgRNAs knock out a stable GFP and include single mismatches in the spacers to mimic off-target effects. b Spearman rank correlation of the SaCas9 specificity model compared with previously reported SaCas9 GUIDE-seq data from a different cell type, U2OS
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