NmeCas9 is an intrinsically high-fidelity genome-editing platform

Abstract

Background: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N₄GATT-3'), for NmeCas9 genome editing in human cells.

Conclusions: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

Keywords: CRISPR; Cas9; Neisseria meningitidis; Off-target; Protospacer adjacent motif; sgRNA.

Fig. 1

NmeCas9 expression and activity in human (HEK293T) cells. a Western blot detection of HA-tagged NmeCas9 in transiently transfected HEK293T cells. Lane 1: cells transfected with SpyCas9 plasmid under the control of the CMV promoter. Lane 2: cells transfected with NmeCas9 plasmid under the control of the elongation factor-1α (EF1α) promoter. Lane 3: cells expressing NmeCas9 and a non-targeting sgRNA (nt-sgRNA), which lacks a complementary site in the human genome. Lane 4: cells expressing NmeCas9 and a sgRNA targeting chromosomal site NTS3. Upper panel: anti-HA western blot. Lower panel: anti-GAPDH western blot as a loading control. b NmeCas9 targeting co-transfected split-GFP reporter with ps9, ps24, and ps25 sites. Plasmid cleavage by SpyCas9 is used as a positive control, and a reporter without a guide-complementary site (No ps: no protospacer) is used as a negative control to define background levels of recombination leading to GFP+ cells. c NmeCas9 programmed independently with different sgRNAs targeting eleven genomic sites flanked by an N₄GATT PAM, detected by T7E1 analysis. Products resulting from NmeCas9 genome editing are denoted by the red dots. d Quantitation of editing efficiencies from three independent biological replicates performed on different days. Error bars indicate ± standard error of the mean (± s.e.m.). e Editing efficiencies for chromosomal target sites as measured by PCR and high-throughput sequencing (Deep sequencing). Data are mean values ± s.e.m. from three biological replicates performed on different days. f Genomic edits with NmeCas9 programmed independently with different guides in different cell lines and using different methods of delivery

Fig. 2

Characterization of functional PAM sequences in human (HEK293T) cells. a Split-GFP activity profile of NmeCas9 cleavage with ps9 sgRNA, with the target site flanked by different PAM sequences. Bars represent mean values ± s.e.m. from three independent biological replicates performed on different days. b T7E1 analysis of editing efficiencies at seven genomic sites flanked by PAM variants, as indicated. Products resulting from NmeCas9 genome editing are denoted by the red dots. c Editing efficiencies for chromosomal target sites with different PAM variants, as in B and C, as measured by deep sequencing analysis. d Quantitation of data from (b), as well as an additional site (NTS31; N₄GACA PAM) that was not successfully edited. Bars represent mean values ± s.e.m. from three independent biological replicates performed on different days

Fig. 3

NmeCas9 and SpyCas9 have comparable editing efficiencies in human (HEK293T) cells when targeting the same chromosomal sites. a Western blot analysis of NmeCas9 and SpyCas9. HEK293T cells were transfected with the indicated Cas9 ortholog cloned in the same plasmid backbone and fused to the same HA epitope tags and NLSs. Top panel: anti-HA western blot (EP, empty sgRNA plasmid). Bottom panel: anti-GAPDH western blot, used as a loading control. Mobilities of protein markers are indicated. b T7E1 analysis of three previously validated SpyCas9 guides targeting the AAVS1 locus, in comparison with NmeCas9 guides targeting nearby AAVS1 sites (mean ± s.e.m., n = 3). c Representative T7EI analyses comparing editing efficiencies at the dual target sites DTS1, DTS3, DTS7, DTS8, and NTS7, using the indicated Cas9/sgRNA combinations. Products resulting from Cas9 genome editing are denoted by the red dots. d Quantitation of data from (c) (mean ± s.e.m., n = 3). Two-tailed paired Student’s T test showed significant difference between NmeCas9 and SpyCas9 editing of DTS1, DTS3, and DTS8 (p < 0.05)

Fig. 4

Bioinformatic and empirical comparison of NmeCas9 and SpyCas9 off-target sites within the human genome. a Genome-wide computational (CRISPRseek) predictions of off-target sites for NmeCas9 (with N₄GN₃ PAMs) and SpyCas9 (with NGG, NGA, and NAG PAMs) with DTS3, DTS7, and DTS8 sgRNAs. Predicted off-target sites were binned based on the number of mismatches (up to six) with the guide sequences. b GUIDE-Seq analysis of off-target sites in HEK293T cells with sgRNAs targeting DTS3, DTS7, and DTS8, using either SpyCas9 or NmeCas9, and with up to 6 mismatches to the sgRNAs. The numbers of detected off-target sites are indicated at the top of each bar. c Numbers of independent GUIDE-Seq reads for the on- and off-target sites for all six Cas9/sgRNA combinations from (b) (SpyCas9, orange; NmeCas9, blue), binned by the number of mismatches with the corresponding guide. d Targeted deep sequencing analysis of editing efficiencies at on- and off-target sites from (a) or (b) with SpyCas9 (left, orange) or NmeCas9 (right, blue). Data for off-target sites are in grey. For SpyCas9, all off-target sites were chosen from (b) based on the highest GUIDE-Seq read counts for each guide (Additional file 10: Table S3). For NmeCas9, in addition to those candidate off-target sites obtained from GUIDE-Seq (c), we also assayed one or two potential off-target sites (designated with the “-CS” suffix) predicted by CRISPRseek as the closest near-cognate matches with permissive PAMs. Data are mean values ± s.e.m. from three biological replicates performed on different days

Fig. 5

Off-target analyses for additional NmeCas9 sgRNAs, targeting sites with consensus and variant PAMs. a Number of GUIDE-Seq reads for the on-target sites, with the PAM sequences for each site indicated underneath. b Number of GUIDE-Seq-detected off-target sites using the Bioconductor package GUIDEseq version 1.1.17 [75] with default settings except that PAM.size = 8, PAM = “NNNNGATT,” min.reads = 2, max.mismatch = 6, allowed.mismatch.PAM = 4, PAM.pattern = “NNNNNNNN$,” BSgenomeName = Hsapiens, txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, orgAnn = org.Hs.egSYMBOL gRNA.size was set to length of the gRNA used, and various number of 0’s were added at the beginning of weights to make the length of weights equal to the gRNA size. For example, for gRNA with length 24, weights = c(0,0,0,0,0, 0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583) for all sixteen sgRNAs used in (a). c Schematic diagrams of NmeCas9 sgRNA/DNA R-loops for the NTS1C (left) and NTS25 (right) sgRNAs, at the GUIDE-Seq-detected on- and off-target sites. Black, DNA residues; boxed nts, PAM; red line, NmeCas9 cleavage site; cyan and purple, mismatch/wobble and complementary nts (respectively) in the NmeCas9 sgRNA guide region; green, NmeCas9 sgRNA repeat nts. d NmeCas9 editing efficiencies at the NTS1C (left) and NTS25 (right) on-target sites, and at the off-target sites detected by GUIDE-Seq from (b), as measured by PCR and high-throughput sequencing. Data are mean values ± s.e.m. from three biological replicates performed on different days. e Comparison of NmeCas9 and SpyCas9 biochemical off-target sites using SITE-Seq analysis

Fig. 6

NmeCas9 guide length requirements in mammalian cells. a Split-GFP activity profile of NmeCas9 cleavage with ps9 sgRNAs bearing spacers of varying lengths (18–24 nt) along with 5′-terminal G residues to enable transcription. Bars represent mean values ± s.e.m. from three independent biological replicates performed on different days. b T7EI analysis of editing efficiencies at the NTS33 genomic target site (with an N₄GATT PAM) with sgRNAs bearing spacers of varying lengths (13–25 nt) with 1–2 5′-terminal G residues. Products resulting from NmeCas9 genome editing are denoted by the red dots. c Quantitation of editing efficiencies (of experiment in b) from three independent biological replicates performed on different days. Error bars indicate ± standard error of the mean (± s.e.m.). d As in (b), but targeting the NTS32 genomic site (with a N₄GCTT PAM). e Quantitation of editing efficiencies (of experiment in d) from three independent biological replicates performed on different days. Error bars indicate ± standard error of the mean (± s.e.m.)

Fig. 7

Guide truncation can suppress off-target editing by NmeCas9. a Editing efficiencies at the NTS1C (on-target, red) and NTS1C-OT1 (off-target, orange) genomic sites, after editing by NmeCas9 and NTS1C sgRNAs of varying lengths, as measured by PCR and high-throughput sequencing. Data are mean values ± s.e.m. from three biological replicates performed on different days. b As in (a), but using sgRNAs perfectly complementary to the NTS1C-OT1 genomic site. Two-tailed paired Student’s T test showed significant difference in on- and off-target editing efficiency as a function of guide truncation. On-target editing (black asterisk) or off-target editing (red asterisk) is compared to the baseline condition GN₂₄, respectively (p < 0.05 is annotated with one asterisk; p < 0.01 is annotated with two asterisks)