Materials and Methods

Genome editing in mammalian cells using the CRISPR type I-D nuclease

MATERIALS AND METHODSVector constructionDetails of all plasmid DNAs used in this study are described in Supplementary Figure S13. PCR amplification for cloning gene fragments was done using PrimeSTAR Max (TaKaRa). Cloning for assembly was done using Quick ligation kit (NEB), NEBuilder HiFi DNA Assembly (NEB), and Multisite gateway Pro (Thermo Fisher Scientific).Gene fragments corresponding to human codon-optimized cas effector genes; cas3d, cas5d, cas6d, cas7d, and cas10d, were synthesized with the SV40 nuclear localization signal (NLS) at their N-termini (gBlocks®) (Integrated DNA Technologies), assembled, and cloned into the pEFs vector (38) separately to yield pEFs-HA-SV40NLS-Cas3d, pEFs-Strept-SV40NLS-Cas5d, pEFs-myc-SV40NLS-Cas6d, pEFs-FLAG-SV40NLS-Cas7d, and pEFs-6xHis-SV40NLS-Cas10d. The tags fused to each Cas protein were as follows; HA-tag to Cas3d, Strep-tag to Cas5d, Myc-tag to Cas6d, FLAG-tag to Cas7d, and 6x His-tag to Cas10d. All cas genes were combined into an all-in-one vector, pEFs-All, by fusing with sequences encoding 2A self-cleavage peptides. pEFs_Cas3d-Cas10d and pEFs_Cas6d-Cas5d-Cas7d expression vectors were also constructed by linking to the 2A self-cleavage peptide. To evaluate different promoters to express Cas5d and Cas6d, the human EFs promoter was replaced with the CAG promoter in pEFs-Strept-SV40NLS-Cas5d, and pEFs-Myc-SV40NLS-Cas6d to yield pCAG-Strept-SV40NLS-Cas5d or pCAG-Myc-SV40NLS-Cas6d. To construct the Cas-expressing vector with bipartite NLS (bpNLS), the DNA fragment (myc-bpNLS-Cas10d-bpNLS-6xHis) was first synthesized (IDT) and cloned into the pEFs vector, resulting in pEFs-Myc-bpNLS-Cas10d-bpNLS-6xHis vector. The cas10d gene fragment in pEFs-Myc-bpNLS-Cas10d-bpNLS-6xHis vector was replaced by cas3d, cas5d, cas6d and cas7d gene fragments, respectively, to yield pEFs-Myc-bpNLS-Cas3d-bpNLS-6xHis, pEFs-Myc-bpNLS-Cas5d-bpNLS-6xHis, pEFs-Myc-bpNLS-Cas7d-bpNLS-6xHis and pEFs-Myc-bpNLS-Cas6d-bpNLS-6xHis. The resulting vectors were used for the construction of pEFs-Myc-bpNLS-Cas3d-FLAG-NLS, pEFs-Myc-bpNLS-Cas5d-FLAG-NLS, pEFs-Myc-bpNLS-Cas6d-FLAG-NLS, pEFs-Myc-bpNLS-Cas7d-FLAG-NLS, pEFs-Myc-bpNLS-Cas10d-FLAG-NLS by replacing bpNLS-6xHis with FLAG-NLS.To construct the mutated Cas10d (H177A) expression vectors, the dCas10d (H177A) fragment was synthesized (IDT) and the wild-type Cas10d fragment in pEFs-Myc-bpNLS-Cas10d-bpNLS-6xHis was replaced by dCas10d (H177A) to yield pEFs-myc-bpNLS-dCas10d (H177A)-bpNLS-6xHis. To construct mutated Cas7 expression vectors with myc-tag and 3xSV40NLS Cas7d (K58A), Cas7d (K62A), Cas7d (K67A), Cas7d (R68A), Cas7d (K69A), Cas7d (T75A), Ca7d (E148Q), Cas7d(F66R/K67A/R68M/K69R), Cas7d (K67S/R68Q), Cas7d (K67A/R68A), Cas7d (K67S/R68Q/K69Q) and Cas7d (F66K/K67S/R68Q), the Cas7d fragment was first cloned into the pEFs2 vector, which has Myc-tag and 3x SV40NLS at its N-terminus to yield pEFs2-Myc-3xSV40NLS-Cas7d. Then, each mutation was introduced by PCR amplification to yield pEFs2-Myc-3xSV40NLS-Cas7d (K58A), pEFs2-Myc-3xSV40NLS-Cas7d (K62A), pEFs2-Myc-3xSV40NLS-Cas7d (K67A), pEFs2-Myc-3xSV40NLS-Cas7d (R68A), pEFs2-Myc-3xSV40NLS-Cas7d (K69A), pEFs2-Myc-3xSV40NLS-Cas7d (T75A), pEFs2-Myc-3xSV40NLS-Ca7d (E148Q), pEFs2-Myc-3xSV40NLS-Cas7d(F66R/K67A/R68M/K69R), pEFs2-Myc-3xSV40NLS-Cas7d (K67S/R68Q), pEFs2-Myc-3xSV40NLS-Cas7d (K67A/R68A), pEFs2-Myc-3xSV40NLS-Cas7d (K67S/R68Q/K69Q) and pEFs2-Myc-3xSV40NLS-Cas7d (F66K/K67S/R68Q). The Cas7d fragment in pEFs2-Myc-3xSV40NLS-Cas7d was replaced with Cas3d, Cas5d, Cas6d, or Cas10d fragment to yield pEFs2-Myc-3xSV40NLS-Cas3d, pEFs2-Myc-3xSV40NLS-Cas5d, pEFs2-Myc-3xSV40NLS-Cas6d, pEFs2-Myc-3xSV40NLS-Cas10d. To construct mutated Cas10d or Cas3d expression vectors that have mutations in ATP-binding domains, Cas3d (D179A), Cas10d (D293A), or Cas10d (K407A) fragments were synthesized (IDT) and the wild-type Cas10d fragment in pEFs2–3xNLS-SV40NLS-Cas10d was replaced by each respective fragment to yield pEFs2-myc-3x SV40NLS-Cas3d (D179A), pEFs2-myc-3x SV40NLS-Cas10d (D293A) and pEFs2-myc-3x SV40NLS-Cas10d (K407A).For crRNA expression, a DNA fragment containing a repeat-spacer-repeat sequence was cloned under the human U6 promoter in pEX-A2J1 (Eurofins Genomics) to yield pAEX-hU6crRNA. To construct pAEX-hU6crRNA_mature, two repeat sequences were replaced by the predicted processed repeat sequences. The mature crRNA was predicted based on a report by Shao and Li (6) and the stability of the secondary structure analyzed using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). For insertion of gRNA sequence, two oligonucleotides containing a target sequence were annealed and cloned into the gRNA expression vector using Golden Gate cloning using the restriction enzyme BsaI (NEB).Luc reporter assay plasmids: for the luc reporter assay, NanoLUxxUC expression vectors were constructed. First, NLUxxUC_Block1 and NLUxxUC_Block2 DNA fragments were synthesized (IDT). NLUxxUC_Block1 includes the first 351 bp of the NanoLUC gene sequence and Multi Cloning Site. An XbaI site was attached to the 5′ end of the NLUxxUC_Block1. NLUxxUC_Block2 includes 465 bp of the 3′ end of the NanoLUC gene. An XhoI site was attached to the 3′ end of the NUxxUC_Block2. These fragments were assembled and replaced with EGxxFP fragment in the pCAG-EGxxFP vector obtained from addgene (#50716) to yield the pCAG-nLUxxUC vector. Each split type of the NLUxxUC reporter (pCAG-nLUxxUC_Block1, pCAG-nLUxxUC_Block2) was constructed by removing the NLUxxUC_Block1 fragment by XhoI and BamHI digestion, or by removing NLUxxUC_Block2 fragment by XbaI and EcoRI digestion from the pCAG-NLUxxUC vector, respectively. Each digested vector was assembled with a Multi Cloning Site to yield pCAG-NLUxxUC_Block1_MCS and pCAG-NLUxxUC_MCS_Block2 vector (Supplementary Figure S13).Cell culture and transfectionHuman embryonic kidney cell line 293T (HEK293T, RIKEN BRC) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), GlutalMAX™ Supplement (Thermo Fisher Scientific), 100 units/ml penicillin and 100 μg/ml streptomycin in a 60 mm dish at 37°C with 5% CO2 incubation. HEK293T cells were seeded into six-well plates (Corning) the day before transfection. Cells were transfected using TurboFect Transfection Reagent (Thermo Fisher Scientific) following the manufacturer's recommended protocol. The repeat-spacer-repeat crRNA was used for the transfection unless otherwise specified. For each well of a six-well plate, a total of 4 μg plasmid DNA, extracted using NucleoSpin® Plasmid Transfection-grade kit (Macherey-Nagel), was used. Transfected cells were harvested after 48 h for mutation analysis.Purification of recombinant Cas proteinsRecombinant Cas10d and Cas3d proteins were expressed in human HEK293T cells and purified as mentioned below. At first, human HEK293T cells were seeded in 150-mm culture dishes a day before transfection. Then, human HEK293T cells were transfected with pEFs-Myc-bpNLS-Cas10d-bpNLS-6xHis or pEFs-Myc-bpNLS-Cas3d-bpNLS-6xHis or pEFs-Myc-bpNLS-Cas10d (H177A)-bpNLS-6xHis vector using Lipofectamine 2000 (Thermo Fisher Scientific). Two days after transfection, the cells were collected and suspended in buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, 10% glycerol). Cells were disrupted by sonication and cell debris was removed by the centrifugation at 12,000 rpm for 15 min at 4°C. The supernatant was collected and mixed with Ni-NTA Agarose (QIAGEN) pre-equilibrated with a buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, 10% glycerol). The mixture was then incubated with rotating in 4°C for 1 h. The Ni-NTA resin was washed with 5 column volumes of buffer 1 (120 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, 10% glycerol, 10 mM imidazole), followed by washing with 5 column volumes of buffer 2 (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, 10% glycerol, 20 mM imidazole). The 6× His-tagged proteins were eluted with 5 column volumes of elution buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, 10% glycerol, 300 mM imidazole). The eluate was then concentrated by ultrafiltration using Apollo device (M & S Techno Systems). The proteins were further purified by gel-filtration chromatography on pre-equilibrated Superdex 200 Increase 10/30GL (Cytiva) using a Biologic DuoFlow 10 system (Bio-Rad). Fractions were analyzed by SDS-PAGE. The fractions with expected proteins were collected and used for in vitro assay.Protein identification by mass spectrometryExcised gel bands were digested with Trypsin (APRO Science) overnight at 37°C. The digested samples were desalted using SPE C-Tip (Nikkyo Technos). The desalted samples were concentrated and resuspended in 0.1% (v/v) formic acid. The recovered peptides were analyzed using Q Exactive Plus (Thermo Fisher Scientific) coupled with a capillary HPLC system (EASY-nLC 1200, Thermo Fisher Scientific) to acquire tandem mass spectrometry (MS/MS) spectra. Data derived from MS/MS spectra were used to search the protein database SWISS-Prot using the MASCOT server (http://www.matrixscience.com) and identify proteins using the program Scaffold viewer (http://www.proteomesoftware.com/products/scaffold). In vitro ssDNA nuclease assayM13mp18 single-strand DNA (ssDNA) (NEB) was used as a substrate for the ssDNA nuclease assay. For the time course study of ssDNA nuclease activity, 700 nM of Cas10d or Cas3d was added to Cut smart buffer (NEB) supplemented with 2 mM ATP, 100 μM CoCl2, 100 μM NiCl2 and 3 nM M13mp18 single-strand DNA) and incubated for 0, 0.5, 1 or 2 h at 37°C. The reaction products were purified by phenol/chloroform extraction and the supernatant analyzed by agarose gel electrophoresis. The DNAs were visualized by GelRed® Nucleic Acid Gel Strain (Biotium). To study the concentration dependence of ssDNA nuclease activity, various amounts of Cas10d (0, 50, 125, 250, 500, 700 μM) were added to the buffer mentioned above. To study the effects of divalent cations, 25 mM EDTA or the following concentrations of each divalent cation were added to a buffer 10 mM Tris–HCl (pH 7.5), 60 mM KCl, 10% glycerol, 3 nM M13mp18 single-strand DNA, 700 nM Cas10d and incubated for 2 h at 37°C; 10, 30, 60 mM of MgCl2 or 50, 100, 150 μM of NiCl2 or 50, 100, 150 μM CoCl2. The range of dication concentration was chosen based on data from Type I-E Cas3 (39). The reaction products were purified by phenol/chloroform extraction and visualized as mentioned above. Experiments were repeated three times independently. In vitro dsDNA nuclease assayPlasmid DNA, pNEB193 (NEB) was linearized by BamHI and used as a substrate for in vitro double-strand DNA (dsDNA) nuclease assay. Linearized DNA (70 ng) was mixed with 700 nM of each Cas protein in Cut smart buffer solution (NEB) supplemented with 2 mM ATP, 100 μM CoCl2 and 100 μM NiCl2, and incubated at 37°C for 0, 0.5, 1 or 2 h. The reaction mixture was subjected to phenol/chloroform extraction. Aqueous fractions were mixed with 6× loading dye and separated by electrophoresis through 1% agarose gel. The DNAs were visualized using GelRed ® Nucleic Acid Gel Strain (Biotium). The experiments were repeated three times independently. In vitro helicase assayOligo DNA (100 nt) labeled with fluorescent dye ATTO532 at the 5′ terminal was used as a ssDNA in this experiment. Partial duplex DNA was prepared by annealing the ATTO532-labeled oligonucleotide DNA (100 nt) to oligo DNA (40 nt) with the complementary sequence. Oligonucleotide DNA sequences are listed in Supplementary Table S1. Annealing was performed by mixing ATTO532-conjugated oligo DNA and complementary oligo DNA at molecular ratio of 1:1.5, with the reaction taking place under the following conditions: 98°C 5 min, cooling to 85°C at a speed of 2°C/s, 85°C 1 min, cooling to 25°C at a speed of 0.1°C/s, 25°C 1 min. The annealed partial duplex DNA (2 μM) was used as a dsDNA for the helicase assay. Cas10d or Cas3d, or both proteins, were added to the reaction buffer (1 mM Tris–HCl, 25 mM KCl, 15% glycerol, 200 μM ATP, 100 μM MgCl2) (20). ssDNA and partial duplex DNA without Cas proteins were used as negative controls. The helicase reaction was performed at 37°C for 2 h in a buffer mentioned above with or without ATP. The reaction was stopped by adding 6× gel loading dye (NEB). The reaction products were analyzed by 8% non-denaturing polyacrylamide electrophoresis. The run was carried out at a constant current of 15 mA for 1 hour. Fluorescence was visualized by Typhoon FLA9500 (Cytiva). The experiments were repeated three times independently.ATPase assayThe ATPase reactions were performed at 37°C in CutSmart reaction buffer (NEB) supplemented with 2 mM ATP, 100 μM CoCl2, 100 μM NiCl2 and 700 nM Cas10d or Cas3d. To investigate the ssDNA-dependent ATPase activity, various amounts of ssDNA (M13mp18) or dsDNA (pNEB193) (0, 0.5, 1, 3, 5, 10 nM) were added to the reaction mixture. Reactions were performed at 37°C for 2 h and stopped by adding 27 mM EDTA. In the time-course study of ATPase activity, each reaction was stopped by adding 27 mM of EDTA solution at fixed time intervals (0, 5, 10, 20, 30, 60 min). The detection of liberated phosphate was performed using BIOMOL® Green Reagent kit (Cosmo Bio Co. Ltd) according to the manufacturer's protocol. The amount of free-liberated phosphate was calculated based on the calibration curve that was established using the phosphate solutions provided by the manufacturer. The amount of liberated phosphate at 0 nM of DNA or at 0 min of reaction time was subtracted as a background from the measured amount of free-liberated phosphate in each experiment. The reaction rate (min–1) was calculated from the slope of the time course. ATPase experiments were performed three times independently.Luc reporter assayHuman HEK293T cells were used for the luc reporter assay. Cells (2.0 × 104 cells/well) were seeded onto 96-well plates (Corning) the day before transfection, and transfected as mentioned above. A total of 200 ng plasmid DNAs including (i) pGL4.53 vector encoding Fluc gene (Promega) used as an internal control, (i) two kinds of pCAG-nLUxxUC vectors (pCAG-nLUxxUC_Block1 and pCAG-nLUxxUC_Block2) interrupted with target DNA fragment (Supplementary Table S3), (iii) plasmid DNAs encoding type I-D CRISPR–Cas components (Supplementary Figure S13, pEFs vectors) and (iv) pAEX-hU6gRNA for the gRNA expression vector were used in each well of a 96-well plate. For the luc reporter assay of EGFP gRNAs, a non-split type LUC vector was used; the pCAG-nLUxxUC vector interrupted with target DNA fragment was used instead of pCAG-nLUxxUC_Block1 and pCAG-nLUxxUC_Block2 vectors. NanoLuc and Fluc luciferase activities were measured 2 days after transfection using the Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega). The NanoLuc/Fluc ratio was calculated for each sample. The NanoLuc/Fluc ratio of the sample transfected with non-targeting gRNAs was used as a control, and the relative activity to the control was calculated for each sample to evaluate the gRNA activity. The experiments were repeated more than three times independently.Western blottingAt 2 days post-transfection, total proteins were extracted from HEK293T cells using RIPA Lysis and Extraction Buffer (Thermo Scientific) supplemented with Protease Inhibitor Cocktail for Use with Mammalian Cell and Tissue Extracts (Nakalai Tesque) according to the manufacturer's protocol. For the isolation of nuclear and cytoplasmic proteins, NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) was used. The extracted proteins were quantified using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). After the quantification of protein concentrations, the samples were mixed with 4× Laemmli Sample buffer (Bio-Rad) and 2.5% β-mercaptoethanol, followed by heat treatment at 95°C for 5 min. The denatured proteins were loaded onto 12% SDS-PAGE gel with Tris–glycine–SDS buffer 0.25 M Tris, 1.92 M glycine, 1% (w/v) SDS and separated by SDS-PAGE for 60 min at 150 V. The proteins were transferred to Immobilon-P Polyvinylidene fluoride (PVDF) membranes (Millipore) in Tris-Glycine-SDS buffer containing 20% methanol for 2 h at 50 V. The blot was washed by TTBS (25 mM Tris, 137 nM NaCl, 2.68 nM KCl) for 5 min three times and blocked at room temperature in Blocking One (Nacalai Tesque) for 60 min. The primary antibody reactions were performed at room temperature for 60 min. After washing the membranes in TTBS for 5 min three times, the secondary antibody, Anti-Mouse IgG (H+L), HRP Conjugate (Promega, 1:10 000 in Blocking one), was added to the membrane. After incubation for 60 min at room temperature, the membrane was washed with TTBS 5 min three times. Signals were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. Images were captured using an ImageQuant LAS4000 mini (GE Healthcare Bioscience). Primary antibodies used in this study are as follows: anti-DDDDK-tag mAb (1:10 000) (MBL, Japan), anti-HA-tag mAb (1:10 000) (MBL), anti-His-tag mAb (1:10 000) (MBL), anti-Myc-tag mAb (1:10 000) (MBL), anti-Strep-tag mAb (1:1000) (MBL), anti-β-actin mAb (1:10 000) (MBL). The experiments were repeated three times independently.ImmunoprecipitationHEK293T cells were transfected using TurboFect Transfection Reagent (Thermo Fisher Scientific) with each Cas expression vector in the following combinations: (i) pEFs-FLAG-SV40NLS-Cas7d with other pEFs2-Myc-3xSV40NLS-Cas-vectors (Cas3d, 5d, 6d, 10d) and crRNA; (ii) pEFs-Myc-bpNLS-Cas3d-FLAG-SV40NLS with other pEFs-Myc-3xSV40NLS-Cas vectors (Cas5d, 6d, 7d, 10d) and crRNA or (iii) pEFs2–3xNLS-SV40NLS-Cas3d and pEFs-Myc-bpNLS-Cas5d-FLAG-SV40NLS or pEFs-Myc-bpNLS-Cas6d-FLAG-SV40NLS or pEFs-Myc-bpNLS-Cas10d-FLAG-SV40NLS or pEFs-FLAG-Cas7d. Protein extraction was performed according to the protocol described in Katoh et al. (40) at 48 h post-transfection. Briefly, the medium was replaced with Lysis buffer 20 mM HEPES, 150 mM NaCl, 0.1% (w/v) Triton X-100, 10% (w/v) glycerol containing Protease Inhibitor Cocktail for Use with Mammalian Cell and Tissue Extracts (Nacalai Tesque) and incubated for 5 min on ice. The cell lysates were mixed by pipetting and transferred to 1.5 ml tubes, then incubated for 15 min on ice. Purification of FLAG-tagged protein was performed using DDDDK-tagged Protein Magnetic Purification Kit (MBL) according to the protocol provided by the manufacturer. The resulting elutes were analyzed by SDS-PAGE and western blotting using following antibodies: anti-His-tag mAb (1:10 000) (MBL), anti-DDDDK-tag mAb (1:10 000) (MBL), anti-β-actin mAb (1:10 000) (MBL), anti-Mouse IgG (H+L) and HRP Conjugate (1:10 000) (Promega). The experiments were repeated three times independently with similar results.DNA deletion analysis by long-range PCRTo detect DNA deletion in HEK293T cells, long-range PCR and cloning of a pool of long PCR products was performed. Genomic DNA was first extracted from HEK293T cells using the Geno Plus™ Genomic DNA Extraction Miniprep System (Viogene-BioTek). Nested PCR was then performed to amplify long-range DNA region specifically. At first, the extracted genome DNA was used as a template for amplification of the target DNA region using several specific kinds of primer sets for long-range PCR (Supplementary Table S5), which were designed to amplify target DNA region of various lengths (3.5–25 kb). The first PCR reactions were performed using KOD ONE Master Mix (TOYOBO) under the following conditions: 35 cycles of 10 s at 98°C, 5 s at 60°C, 50 s (amplicon: <10 kb) or 150 s (amplicon: 10–15 kb) or 200 s (amplicon: 15–20 kb) at 68°C. These PCR products were then diluted 100–10 000 times and used as a template for nested PCR. The nested PCR was performed under same conditions mentioned above. The PCR products were separated by electrophoresis on 1% agarose gel and visualized by staining with GelRed™ Nucleic Acid Gel Stain (Biotium). Nested PCR products were pooled and purified using Monofas® DNA purification Kit I (GL Sciences). The purified mixture of PCR products was cloned into pMD20-T vector using a Mighty TA-cloning Kit (TaKaRa). The 119 clones for AAVS- GTC_70–107 (+) and 20 clones for hEMX1- GTT_998–1036 (–) were picked up and Sanger sequenced using M13 Uni and M13 RV primers. Sanger sequencing results were analyzed using BLASTN search and ClustalW program to identify DNA deletions.Mutation analyses in short-range PCR productsTo evaluate mutations introduced in transfected human cells, a region of about 400 bp surrounding the target locus of gRNA was amplified by short-range PCR using a PCR kit as described above. In heteroduplex mobility assay (HMA), the PCR fragments were analyzed directly using a microchip electrophoresis system with MCE202 MultiNA (Shimazu). PCR amplicons were also cloned into the TA cloning vector (TaKaRa) to determine their sequences by the Sanger method. All primers used for short-range PCRs used in the mutation analyses are listed in Supplementary Table S4.Multiple alignmentA multiple alignment of the HD domain of Cas3 and Cas10d protein sequences was constructed by using ClustalX (http://www.clustal.org/clustal2/), with pairwise parameters as follows: gap opening = 10, gap extend = 0.1 and protein weight matrix = identity matrix; multiple parameters as gap opening = 10, gap extension = 0.2, delay divergent sequence (%) = 15, protein weight matrix = identity matrix. Gene accession numbers of protein sequences used for the alignment were MaCas10d: M. aeruginosa ({"type":"entrez-protein","attrs":{"text":"WP_002791883.1","term_id":"488879658","term_text":"WP_002791883.1"}}WP_002791883.1), AcCas3a: Anabaena cylindrica ({"type":"entrez-protein","attrs":{"text":"WP_081593764.1","term_id":"1174920544","term_text":"WP_081593764.1"}}WP_081593764.1), BtCas3a: Bacillus thuringiensis ({"type":"entrez-protein","attrs":{"text":"WP_000506550.1","term_id":"446428695","term_text":"WP_000506550.1"}}WP_000506550.1), HhCas3a Halobacteroides halobius ({"type":"entrez-protein","attrs":{"text":"WP_015326643.1","term_id":"505139541","term_text":"WP_015326643.1"}}WP_015326643.1), BcCas3b: Bacillus cytotoxicus ({"type":"entrez-nucleotide","attrs":{"text":"NC_009674","term_id":"152973854","term_text":"NC_009674"}}NC_009674), LrCas3b: Lactobacillus ruminis ({"type":"entrez-nucleotide","attrs":{"text":"NC_015975","term_id":"347524522","term_text":"NC_015975"}}NC_015975), SaCas3b: Sulfobacillus acidophilus ({"type":"entrez-nucleotide","attrs":{"text":"NC_016884","term_id":"379005799","term_text":"NC_016884"}}NC_016884), ErCas3c: Eubacterium rectale ({"type":"entrez-nucleotide","attrs":{"text":"NC_021044","term_id":"479213596","term_text":"NC_021044"}}NC_021044), LfCas3c: Lactobacillus fermentum ({"type":"entrez-nucleotide","attrs":{"text":"NC_017465","term_id":"385811786","term_text":"NC_017465"}}NC_017465), XoCas3c: Xanthomonas oryzae ({"type":"entrez-nucleotide","attrs":{"text":"NC_007705","term_id":"84621657","term_text":"NC_007705"}}NC_007705), EcCas3e: Escherichia coli ({"type":"entrez-protein","attrs":{"text":"EIG78940.1","term_id":"386137778","term_text":"EIG78940.1"}}EIG78940.1), PaCas3e: Pseudomonas aeruginosa ({"type":"entrez-nucleotide","attrs":{"text":"NC_021577","term_id":"514407635","term_text":"NC_021577"}}NC_021577), TfCas3e: Thermobifida fusca ({"type":"entrez-protein","attrs":{"text":"AAZ55629.1","term_id":"71915727","term_text":"AAZ55629.1"}}AAZ55629.1), EcCas3f: Escherichia coli ({"type":"entrez-nucleotide","attrs":{"text":"NC_008563","term_id":"117622295","term_text":"NC_008563"}}NC_008563), LpCas3f: Legionella pneumophila ({"type":"entrez-nucleotide","attrs":{"text":"NC_006366","term_id":"54292907","term_text":"NC_006366"}}NC_006366), YpCas3f: Yersinia pestis ({"type":"entrez-nucleotide","attrs":{"text":"NC_017168","term_id":"384137007","term_text":"NC_017168"}}NC_017168).Molecular dynamics simulations of the in silico model of cascade complexes of type I-D CRISPR–Cas, type I-E CRISPR–Cas and type I-F CRISPR–CasThe Cas7d structure in the type I-D CRISPR–Cas Cascade complex was predicted using a homology modeling method, MODELLER (41), and an in silico structure prediction method, ROSETTA (42). To check the stability of the in silico model structure of Cas7d, 100 ns molecular dynamics (MD) simulations were performed. The type I-D Cascade complex structure was constructed using five in silico models for Cas7d, and a crRNA. The structure of the Type I-E CRISPR Cascade complex (14) was used as the reference conformation for the type I-D Cascade.The MD simulations of the in silico model structure of the type I-D Cascade complex, the Type I-E CRISPR Cascade complex (PDB ID:5H9F), and the Type I-F CRISPR Cascade complex (PDB ID: 5UZ9) (43) were performed using GROMACS software (44) with Amber14sb-permbsc1 force field (45). In the case of the Type I-F structure, we removed the anti-CRISPR molecules, before simulation. Concentrations of K+ and Cl– were set to 150 mM, and the temperature and pressure in the system were set to 300 K and 1 bar, respectively. The MD simulation times were 1.5 μs for Type I-E, and 1.0 μs for Type I-F CRISPR systems. The MD simulation of the in silico model system of type I-D was performed for 100 ns. VMD software (46) was used to display the structures of proteins, DNA, and crRNA.

Article TitleGenome editing in mammalian cells using the CRISPR type I-D nuclease

Abstract

Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.


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