(A) Schematic of the GFP-activation assay. A protospacer flanked by an 8 bp random sequence is inserted between the ATG start codon and GFP-coding sequence, resulting in a frameshift mutation. The …
The maps of all plasmids used in the study for Figure 1.
The amino acid sequences of 29 selected Nme1Cas9 orthologs were aligned by Vector NTI. Nme1Cas9, Nme2Cas9, and Nme3Cas9 were used as reference and shown in green.
The Nme1Cas9 orthologs contained aspartate (A), histidine (B), or asparagine (C) residues corresponding to the Nme1Cas9 H1024. The PI domains were aligned by Vector NTI. Amino acids crucial for PAM …
(A) Alignment of direct repeat sequences for Nme1Cas9 orthologs is shown. (B) Alignment of tracrRNAs for Nme1Cas9 orthologs. Sequence alignment revealed that direct repeats and the 5’ end of …
In silico co-folding of the crRNA direct repeat and putative tracrRNA shows stable secondary structure and complementarity between the two RNAs.
HEK293T cells without Cas9 transfection were used as a negative control (NC).
Source data for Figure 1—figure supplement 5.
(A) Example of indel sequences measured by deep sequencing for Nsp2Cas9. The GFP coding sequences are shown in green; an 8 bp random sequence is shown in orange; black dashes indicate deleted bases; …
The number of unique PAM sequences and the median coverage of every individual PAM variant for the Figure 2B and C.
(A) PAM wheels for Nme1Cas9 orthologs containing an aspartate residue corresponding to the Nme1Cas9 H1024. PAM positions in the screening assay are shown on the bottom right. (B) PAM wheels for …
(A) Calculated structural model of Bdecas9. The amino acid near the 5 position of the NTS is histidine. Histidine’s side chain forms a potential hydrogen bond with the 6-hydroxyl group of guanine, …
(A) Schematic of Cas9 and sgRNA expression constructs. U1A: U1A promoter; pA: polyA; NLS: nuclear localization signal; HA: HA tag. (B) Protein expression levels of Nsp2Cas9 and Nme2Cas9 were …
Source data for Figure 3C and D.
Source data for Figure 2B.
A single G5 site on the GRIN2B gene was targeted by sgRNAs with spacer lengths varying from 18 to 26 nt.
Source data for Figure 3—figure supplement 1.
Nsp2Cas9 enables editing in HeLa (A), HCT116 (B), A375 (C), SH-SY5Y (D) and mouse N2a cells (E). Data represent mean ± SD (n=3).
Source data for Figure 3—figure supplement 2A-D.
(A) Schematic of the GFP-activation reporter construct for testing engineered Nsp2Cas9 activity. The protospacer sequence is shown below. (B) GFP-positive cells induced by the engineered Nsp2Cas9 …
Source data for Figure 3—figure supplement 3B.
(A) Schematic of Cas9 and sgRNA expression constructs. U1A: U1A promoter; pA: polyA; NLS: nuclear localization signal; HA: HA tag. (B) NarCas9 enables genome editing in HEK293T cells. Data represent …
Source data for Figure 3—figure supplement 4B.
Source data for Figure 3—figure supplement 4C.
(A) Schematic diagram of chimeric Cas9 nucleases based on Nsp2Cas9. PI domain of Nsp2Cas9 was replaced with the PI domain of SmuCas9. (B) Sequence logos and (C) PAM wheel diagrams indicate that …
Source data for Figure 4D and E.
(A) Nsp2-SmuCas9 and Nme2-SmuCas9 could induce GFP expression. Reporter cells without Cas9 transfection are used as a negative control. Scale bar: 250 μm. (B) Sequence logo and (C) PAM wheel diagram …
Source data for Figure 4—figure supplement 1D.
(A) An alignment of the protein tertiary structures of Nsp2-NarCas9 and Nme1Cas9-sgRNA-dsDNA complexes. Blue represents the Nme1Cas9 protein, and green represents the PI domain of NarCas9, PID: PAM …
(A) Calculated structural models of Nme2-NarCas9 and Nme2-SmuCas9 chimeras. In the inactive Nme2-NarCas9 chimera (magenta), R1052 will crash with the DNA strand, leading to a failure of binding with …
(A) Schematic of Cas9 and sgRNA expression constructs. U1A: U1A promoter; pA: polyA; NLS: nuclear localization signal; HA: HA tag. (B) Protein expression levels of Nsp2Cas9, Nsp2-SmuCas9, and SpCas9 …
Source data for Figure 5B.
Source data for the Figure 5C and D.
(A) Analysis of Nsp2Cas9 and Nme2Cas9 specificity with a GFP-activation assay. A panel of sgRNAs with dinucleotide mutations (red) is shown below. The editing efficiencies reflected by ratio of …
Source data for the Figure 6A.
(A) Analysis of Nsp2-SmuCas9 specificity with a GFP-activation assay. A panel of sgRNAs with dinucleotide mutations (red) is shown below. The editing efficiencies reflected by ratio of GFP-positive …
Source data for the Figure 7A.
UniProt ID | Host strain | Name | Length (aa) | Identity toNme1Cas9 (%) |
---|---|---|---|---|
A0A011P7F8 | Mannheimia granulomatis | MgrCas9 | 1,049 | 65.5 |
A0A0A2YBT2 | Gallibacterium anatis IPDH697-78 | GanCas9 | 1,035 | 59.7 |
A0A0J0YQ19 | Neisseria arctica | NarCas9 | 1,070 | 70.4 |
A0A1T0B6J6 | [Haemophilus felis] | HfeCas9 | 1,058 | 65.3 |
A0A1X3DFB7 | Neisseria dentiae | NdeCas9 | 1,074 | 66.4 |
A0A263HCH5 | Actinobacillus seminis | AseCas9 | 1,059 | 66 |
A0A2M8S290 | Conservatibacter flavescens | CflCas9 | 1,063 | 64.2 |
A0A2U0SK41 | Pasteurella langaaensis DSM 22999 | PlaCas9 | 1,056 | 63.9 |
A0A356E7S3 | Pasteurellaceae bacterium | PstCas9 | 1,076 | 63 |
A0A369Z1C7 | Haemophilus parainfluenzae | Hpa1Cas9 | 1,056 | 64.8 |
A0A369Z3K3 | Haemophilus parainfluenzae | Hpa2Cas9 | 1,054 | 65.2 |
A0A377J007 | Haemophilus pittmaniae | HpiCas9 | 1,053 | 65.2 |
A0A378UFN0 | Bergeriella denitrificans (Neisseria denitrificans) | BdeCas9 | 1,069 | 68.8 |
A0A379B6M0 | Pasteurella mairii | PmaCas9 | 1,061 | 63.1 |
A0A379CB86 | Phocoenobacter uteri | PutCas9 | 1,059 | 63 |
A0A380MYP0 | Suttonella indologenes | SinCas9 | 1,071 | 67.8 |
A0A3N3EE71 | Neisseria animalis | Nan1Cas9 | 1,074 | 66.6 |
A0A3S4XT82 | Neisseria animaloris | Nan2Cas9 | 1,078 | 65.4 |
A0A420XER8 | Otariodibacter oris | OorCas9 | 1,058 | 64.4 |
A0A448K7T0 | Pasteurella aerogenes | PaeCas9 | 1,056 | 68.2 |
A0A4S2QB06 | Rodentibacter pneumotropicus | RpnCas9 | 1,055 | 63.7 |
A0A4Y9GBC9 | Neisseria sp. WF04 | Nsp2Cas9 | 1,067 | 59.6 |
A6VLA7 | Actinobacillus succinogenes (strain ATCC 55618/DSM 22257/130Z) | AsuCas9 | 1,062 | 64.6 |
C5S1N0 | Actinobacillus minor NM305 | AmiCas9 | 1,056 | 67.7 |
E0F2V7 | Actinobacillus pleuropneumoniae serovar 10 str. D13039 | ApsCas9 | 1,054 | 65.4 |
F2B8K0 | Neisseria bacilliformis ATCC BAA-1200 | NbaCas9 | 1,077 | 66.5 |
J4KDT3 | Haemophilus sputorum | HspCas9 | 1,052 | 65.2 |
V9H606 | Simonsiella muelleri ATCC 29453 | SmuCas9 | 1,063 | 62.2 |
W0Q6X6 | Mannheimia sp. USDA-ARS-USMARC-1261 | MspCas9 | 1,047 | 65.7 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Homo sapiens) | AAVS1 | GenBank | HGNC:HGNC:22 | |
Gene (Homo sapiens) | VEGFA | GenBank | HGNC:HGNC:12680 | |
Gene (Homo sapiens) | EMX1 | GenBank | HGNC:HGNC:3340 | |
Gene (Homo sapiens) | GRIN2B | GenBank | HGNC:HGNC:4586 | |
Recombinant DNA reagent | Instant Sticky-end Ligase Master Mix | NEB | Catalog #: M0370S | |
Recombinant DNA reagent | T4 DNA ligase | NEB | Catalog #: M0202S | |
Cell line (Homo-sapiens) | HEK293T (normal, Adult) | ATCC | CRL-3216 | |
Cell line (Homo-sapiens) | HeLa | ATCC | CRM-CCL-2 | |
Cell line (Homo-sapiens) | SH-SY5Y | ATCC | CRL-2266 | |
Cell line (Homo-sapiens) | A375 (normal, Adult) | ATCC | CRL-1619 | |
Cell line (Homo-sapiens) | HCT116 (adult male) | ATCC | CCL-247 | |
Cell line (Mus musculus) | N2a cells (mouse neuroblasts) | ATCC | CCL-131 | |
Antibody | Anti-HA (rabbit polyclonal) | abcom | abcam: ab137838; RRID:AB_262051 | (1:1000) |
Antibody | Anti-GAPDH (rabbit polyclonal) | Cell Signaling | Cell Signaling:#3683; RRID:AB_307275 | (1:1000) |
Sequence-based reagent | dsODN-F | This paper | dsODN oligo primer | 5ʹ- P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T - 3ʹ |
Sequence-based reagent | dsODN-R | This paper | dsODN oligo primer | 5ʹ- P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C –3ʹ |
Sequence-based reagent | P5_index_F | This paper | PCR primers | AATGATACGGCGACCACCGAGATCTACACTGAACCTTA CACTCTTTCCCTACACGAC |
Sequence-based reagent | Nuclease_off_+_G SP | This paper | PCR primers | GGATCTCGACGCTCTCCCTATACCGTTATTAACATATGACA |
Sequence-based reagent | Nuclease_off_- _GSP1 | This paper | PCR primers | GGATCTCGACGCTCTCCCTGTTTAATTGAGTTGTCATATGTTAATAAC |
Sequence-based reagent | P5_2 | This paper | PCR primers | AATGATACGGCGACCACCGAGATCTACAC |
Sequence-based reagent | Nuclease_off _+_GSP2 | This paper | PCR primers | CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTGACTGGAGT TCAGACGTGTGCTCTTCCGATCTACATATGACAACTCAATTAAAC |
Sequence-based reagent | Nuclease_off _- _GSP2 | This paper | PCR primers | CAAGCAGAAGACGGCATACGAGATCTAGTACGGTGAC TGGAGTCCTCTCTATGGGCAGTCGGTGATTTGAGTTG TCATATGTTAATAACGGTA |
Software, algorithm | FlowJo software | FlowJo VX | ||
Software, algorithm | CorelDRAW 2020 software | CorelDRAW 2020 | ||
Software, algorithm | Vector NTI software | Vector NTI | ||
Software, algorithm | GraphPad Prism software | GraphPad Prism 7 (https://graphpad.com) | RRID:SCR_015807 | Version 7.0.0 |
Chemical compound, drug | SYBR Gold nucleic acid stain | Thermo Fisher Scientific | Thermo Fisher Scientific: S11494 |
The Cas9 ID and human codon–optimized Cas9 gene.
The file contains the Cas9 ID, tracrRNA, and amino acid sequences of Nme1Cas9 orthologs used in this study. The human codon-optimized Cas9 genes were synthesized. The primers used for the chimera’s construction were also listed in this file.
The single-guide RNA sequence.
The single-guide RNA sequence for Nme1Cas9 orthologs and chimeras used in this study.
Primers used in this study.
A list of oligonucleotide pairs and primers used for deep sequencing.
Target sites used in this study.
A list of the endogenous target sites of human and mouse and their downstream PAM. PAM, protospacer adjacent motif.
Underlying values for all reported summary statistics.
Raw data from all reported summary statistics.