Closely related type II-C Cas9 orthologs recognize diverse PAMs

  1. Jingjing Wei
  2. Linghui Hou
  3. Jingtong Liu
  4. Ziwen Wang
  5. Siqi Gao
  6. Tao Qi
  7. Song Gao
  8. Shuna Sun  Is a corresponding author
  9. Yongming Wang  Is a corresponding author
  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, China
  2. State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, China
  3. Children’s Hospital of Fudan University, National Children’s Medical Center, China
  4. Shanghai Engineering Research Center of Industrial Microorganisms, China
7 figures, 2 tables and 6 additional files

Figures

Figure 1 with 5 supplements
Screening of Nme1Cas9 orthologs activities through a GFP- activation assay.

(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 …

Figure 1—figure supplement 1
Phylogenetic tree of the selected Nme1Cas9 orthologs.

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.

Figure 1—figure supplement 2
Alignment of the PI domain of Nme1Cas9 orthologs.

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 …

Figure 1—figure supplement 3
The alignment of direct repeats and tracrRNAs of Nme1Cas9 orthologs.

(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 …

Figure 1—figure supplement 4
Single-guide RNA (sgRNA) scaffolds of Nme1Cas9 orthologs.

In silico co-folding of the crRNA direct repeat and putative tracrRNA shows stable secondary structure and complementarity between the two RNAs.

Figure 1—figure supplement 5
Protein expression levels of Nme2Cas9 orthologs were analyzed by western blot.

HEK293T cells without Cas9 transfection were used as a negative control (NC).

Figure 2 with 2 supplements
PAM analysis for each Cas9 nuclease.

(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; …

Figure 2—source data 1

The number of unique PAM sequences and the median coverage of every individual PAM variant for the Figure 2B and C.

https://cdn.elifesciences.org/articles/77825/elife-77825-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
PAM wheels for Nme1Cas9 orthologs.

(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 …

Figure 2—figure supplement 2
The specificity between amino acids and bases in calculated structural models.

(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, …

Figure 3 with 4 supplements
Nsp2Cas9 enables editing in HEK293T cells.

(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 …

Figure 3—figure supplement 1
The effect of spacer length on the efficiency of Nsp2Cas9 editing.

A single G5 site on the GRIN2B gene was targeted by sgRNAs with spacer lengths varying from 18 to 26 nt.

Figure 3—figure supplement 2
Nsp2Cas9 enables editing in different mammalian cells.

Nsp2Cas9 enables editing in HeLa (A), HCT116 (B), A375 (C), SH-SY5Y (D) and mouse N2a cells (E). Data represent mean ± SD (n=3).

Figure 3—figure supplement 3
Rational engineering of Nsp2Cas9.

(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 …

Figure 3—figure supplement 4
NarCas9 enables genome editing in mammalian cells.

(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 …

Figure 4 with 3 supplements
Characterization of Nsp2-SmuCas9 for genome editing.

(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 …

Figure 4—figure supplement 1
Test of 4 chimeric Cas9 activity through a GFP-activation assay.

(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 …

Figure 4—figure supplement 2
Structure of the fully complementary Cas9-sgRNA-dsDNA complex in a catalytic state.

(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 …

Figure 4—figure supplement 3
Calculated structural models of Nme2-NarCas9 and Nme2-SmuCas9 chimeras.

(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 …

Comparison of indel efficiency between Nsp2Cas9, Nsp2-SmuCas9, and SpCas9.

(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 …

Analysis of Nsp2Cas9 specificity.

(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 …

Analysis of Nsp2-SmuCas9 specificity.

(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 …

Tables

Table 1
Nme1Cas9 orthologs selected from the UniProt database.
UniProt IDHost strainNameLength (aa)Identity toNme1Cas9 (%)
A0A011P7F8Mannheimia granulomatisMgrCas91,04965.5
A0A0A2YBT2Gallibacterium anatis IPDH697-78GanCas91,03559.7
A0A0J0YQ19Neisseria arcticaNarCas91,07070.4
A0A1T0B6J6[Haemophilus felis]HfeCas91,05865.3
A0A1X3DFB7Neisseria dentiaeNdeCas91,07466.4
A0A263HCH5Actinobacillus seminisAseCas91,05966
A0A2M8S290Conservatibacter flavescensCflCas91,06364.2
A0A2U0SK41Pasteurella langaaensis DSM 22999PlaCas91,05663.9
A0A356E7S3Pasteurellaceae bacteriumPstCas91,07663
A0A369Z1C7Haemophilus parainfluenzaeHpa1Cas91,05664.8
A0A369Z3K3Haemophilus parainfluenzaeHpa2Cas91,05465.2
A0A377J007Haemophilus pittmaniaeHpiCas91,05365.2
A0A378UFN0Bergeriella denitrificans (Neisseria denitrificans)BdeCas91,06968.8
A0A379B6M0Pasteurella mairiiPmaCas91,06163.1
A0A379CB86Phocoenobacter uteriPutCas91,05963
A0A380MYP0Suttonella indologenesSinCas91,07167.8
A0A3N3EE71Neisseria animalisNan1Cas91,07466.6
A0A3S4XT82Neisseria animalorisNan2Cas91,07865.4
A0A420XER8Otariodibacter orisOorCas91,05864.4
A0A448K7T0Pasteurella aerogenesPaeCas91,05668.2
A0A4S2QB06Rodentibacter pneumotropicusRpnCas91,05563.7
A0A4Y9GBC9Neisseria sp. WF04Nsp2Cas91,06759.6
A6VLA7Actinobacillus succinogenes (strain ATCC 55618/DSM 22257/130Z)AsuCas91,06264.6
C5S1N0Actinobacillus minor NM305AmiCas91,05667.7
E0F2V7Actinobacillus pleuropneumoniae serovar 10 str. D13039ApsCas91,05465.4
F2B8K0Neisseria bacilliformis ATCC BAA-1200NbaCas91,07766.5
J4KDT3Haemophilus sputorumHspCas91,05265.2
V9H606Simonsiella muelleri ATCC 29453SmuCas91,06362.2
W0Q6X6Mannheimia sp. USDA-ARS-USMARC-1261MspCas91,04765.7
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene
(Homo sapiens)
AAVS1GenBankHGNC:HGNC:22
Gene
(Homo sapiens)
VEGFAGenBankHGNC:HGNC:12680
Gene
(Homo sapiens)
EMX1GenBankHGNC:HGNC:3340
Gene
(Homo sapiens)
GRIN2BGenBankHGNC:HGNC:4586
Recombinant DNA reagentInstant Sticky-end Ligase Master MixNEBCatalog #: M0370S
Recombinant DNA reagentT4 DNA ligaseNEBCatalog #: M0202S
Cell line (Homo-sapiens)HEK293T
(normal, Adult)
ATCCCRL-3216
Cell line (Homo-sapiens)HeLaATCCCRM-CCL-2
Cell line (Homo-sapiens)SH-SY5YATCCCRL-2266
Cell line (Homo-sapiens)A375
(normal, Adult)
ATCCCRL-1619
Cell line (Homo-sapiens)HCT116
(adult male)
ATCCCCL-247
Cell line (Mus musculus)N2a cells
(mouse neuroblasts)
ATCCCCL-131
AntibodyAnti-HA (rabbit polyclonal)abcomabcam: ab137838; RRID:AB_262051(1:1000)
AntibodyAnti-GAPDH (rabbit polyclonal)Cell SignalingCell Signaling:#3683; RRID:AB_307275(1:1000)
Sequence-based reagentdsODN-FThis paperdsODN oligo primer5ʹ- P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T -
Sequence-based reagentdsODN-RThis paperdsODN oligo primer5ʹ- P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C
–3ʹ
Sequence-based reagentP5_index_FThis paperPCR primersAATGATACGGCGACCACCGAGATCTACACTGAACCTTA
CACTCTTTCCCTACACGAC
Sequence-based reagentNuclease_off_+_G
SP
This paperPCR primersGGATCTCGACGCTCTCCCTATACCGTTATTAACATATGACA
Sequence-based reagentNuclease_off_-
_GSP1
This paperPCR primersGGATCTCGACGCTCTCCCTGTTTAATTGAGTTGTCATATGTTAATAAC
Sequence-based reagentP5_2This paperPCR primersAATGATACGGCGACCACCGAGATCTACAC
Sequence-based reagentNuclease_off
_+_GSP2
This paperPCR primersCAAGCAGAAGACGGCATACGAGATTCGCCTTAGTGACTGGAGT
TCAGACGTGTGCTCTTCCGATCTACATATGACAACTCAATTAAAC
Sequence-based reagentNuclease_off _-
_GSP2
This paperPCR primersCAAGCAGAAGACGGCATACGAGATCTAGTACGGTGAC
TGGAGTCCTCTCTATGGGCAGTCGGTGATTTGAGTTG
TCATATGTTAATAACGGTA
Software, algorithmFlowJo softwareFlowJo VX
Software, algorithmCorelDRAW 2020 softwareCorelDRAW 2020
Software, algorithmVector NTI softwareVector NTI
Software, algorithmGraphPad Prism softwareGraphPad Prism 7 (https://graphpad.com)RRID:SCR_015807Version 7.0.0
Chemical compound, drugSYBR Gold nucleic acid stainThermo Fisher ScientificThermo Fisher Scientific: S11494

Additional files

Supplementary file 1

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.

https://cdn.elifesciences.org/articles/77825/elife-77825-supp1-v2.xlsx
Supplementary file 2

The single-guide RNA sequence.

The single-guide RNA sequence for Nme1Cas9 orthologs and chimeras used in this study.

https://cdn.elifesciences.org/articles/77825/elife-77825-supp2-v2.xlsx
Supplementary file 3

Primers used in this study.

A list of oligonucleotide pairs and primers used for deep sequencing.

https://cdn.elifesciences.org/articles/77825/elife-77825-supp3-v2.xlsx
Supplementary file 4

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.

https://cdn.elifesciences.org/articles/77825/elife-77825-supp4-v2.xlsx
Supplementary file 5

Underlying values for all reported summary statistics.

Raw data from all reported summary statistics.

https://cdn.elifesciences.org/articles/77825/elife-77825-supp5-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/77825/elife-77825-transrepform1-v2.docx

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