Hifi cas9
Manufactured by Integrated DNA Technologies
Sourced in United States
The HiFi Cas9 is a highly specific and accurate CRISPR-associated protein from Integrated DNA Technologies. It is designed for efficient genome editing with reduced off-target effects.
Automatically generated - may contain errors
Lab products found in correlation
Tracrrna, by Integrated DNA Technologies (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Crrna, by Integrated DNA Technologies (1 mentions)
Sgrna, by TriLink (1 mentions)
4d nucleofector, by Lonza (1 mentions)
Idt duplex buffer, by Integrated DNA Technologies (1 mentions)
Nucleofector solution, by Lonza (1 mentions)
Immunocult human cd3 cd28 cd2, by STEMCELL (1 mentions)
P3 solution, by Lonza (1 mentions)
Duplex buffer, by Integrated DNA Technologies (1 mentions)
Megaruptor 2, by Diagenode (1 mentions)
Sqk lsk109 kit, by Oxford Nanopore (1 mentions)
Minion system, by Oxford Nanopore (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Bluepippin system, by Sage Science (1 mentions)
Nucleospin plant 2 kit, by Macherey-Nagel (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
8 protocols using hifi cas9
1
Constructing gRNA/Cas9 Protein RNP Complex
2
CRISPR-Cas9 Editing of CCR5 in HSPCs
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An sgRNA targeting CCR5 exon 3 (sequence; 5ʹ-GCAGCATAGTGAGCCCAGAA-3ʹ) was purchased from TriLink Biotechnologies (San Diego, CA, USA) with the chemical modification 2′-O-methyl-3ʹ-phosphorothioate25 (link). Cas9 and Hifi Cas9 were purchased from Integrated DNA Technologies (IDT, San Jose, CA, USA Catalog #1081058 and #1081060). The editing procedure was performed as follows: sgRNA and Cas9 protein were complexed at a molar ration of 1:2.5 (sgRNA:Cas9) at room temperature for 5 min. The RNP was electroporated into human CD34+ HSPCs 48 h after thawing using the Lonza 4D nucleofector with the following conditions: pulse code: DZ100; cell density: 1 × 106 cells in 100 µl; [Cas9]: 30 µg; [sgRNA]: 15 µg. Following electroporation, cells were immediately rescued with HSPC culture media pre-warmed to 37 °C. rAAV6 was applied to cells at a MOI of 10,000–20,000. The frequency of indel formation was quantified using Tracking Indels by Decomposition (TIDE)70 (link). CCR5 expression was quantified by flow cytometry using anti-human CCR5-APC antibody (BD Biosciences, #556903).
3
CRISPR/Cas9 Knockout of TSPO in C20 Cells
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CRISPR/Cas9 genome editing experiments in C20 cells were performed with lipofection of high-fidelity (hiFi) Cas9 (Integrated DNA Technologies, Inc.) ribonucleoprotein (RNP) complex as described previously (Vakulskas et al. 2018; DOI 10.1038/s41591-018-0137-0). Briefly, guide RNAs were designed using web tool (https://zlab.bio/guide-design-resources ) (access on 28.02.2017), and combined with tracrRNA (Integrated DNA Technologies, Inc., Coralville, IA, USA) in equal amounts in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM Potassium Acetate) at 1 μM concentration by heating the oligos to 95 °C for 5 min and slowly cooling to room temperature. RNP complexes were formed by the addition of HiFi Cas9 enzyme, and C20 cells were transfected using RNAiMax (Thermo Fischer Scientific, Dreieich, Germany) transfection reagent. After 2 days the cells were singled out for clonal selection. Single clones were collected, and the successful knockout was confirmed by western blot using anti rabbit TSPO antibody and sequencing of genomic DNA.
4
Chromosome-specific CRISPR-Cas9 Nucleofection
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Guide RNAs (gRNAs) recognizing chromosomes 1p and 19q sequences were designed using the CRISPick website (https://portals.broadinstitute.org/gppx/crispick/public ). Two gRNAs for each chromosome arm were chemically synthesized (Synthego) (Supplementary Table S1 ). Nucleofection of ribonucleoprotein (RNP) complexes consisting of gRNAs and Cas9 was performed as described with modifications.19 (link) In brief, 400 pmol of HiFi Cas9 (Integrated DNA Technologies) and 500 pmol sgRNA were mixed and incubated at room temperature for 10 minutes. 1 × 106 cells were washed with Opti-MEM (ThermoFisher Scientific) and resuspended in 100 μl Nucleofector Solution (Lonza), gently mixed with pre-assembled RNPs, and electroporated with appropriate programs using Nucleofector 2b or 4d (Lonza). The nucleofection program used for HEK 293T, LN-229, and U-251 cells was Q-001, X-009, and DS-138, respectively.
5
CRISPR-Cas9 Targeting of LDLR Gene
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The following sequences are the target sequence of each gRNA in the LDLR gene.
CGTGGCAGCGAAACTCGTCC.
ACGAGTTTCGCTGCCACGAT.
CCGAGCCATCTTCGCAGTCG.
AACGAGTTCCAGTGCGAAGA.
TGCATTTCCCGTCTTCGCAC.
GCTGAAGTCCCCGGATTTGC.
6
Efficient Gene Editing of Activated T Cells
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When using frozen CD4+ T cells, cells were thawed in T cell media overnight with 50U/mL IL-2 before resuspending in P3 solution (Lonza) while freshly isolated CD4+ T cells were directly resuspended in P3 solution at 106 cells/20µL and nucleofected with 54.9 pmol of Hi-Fi Cas9 (Integrated DNA Technologies) and 33 pmol of each sgRNA (Synthego) using the 4D-16 well strips using program EO-115 (Lonza). No differences were observed in editing efficiency or downstream characterization between fresh and frozen starting T cells (data not shown). Cells were plated directly into T cell media containing 50U/mL IL-2 after nucleofection. After 2 days, cells were stimulated using 10µL/1mL of ImmunoCult Human CD3/CD28/CD2 (STEMCELL Technologies). After 3 days, media was replaced with T cell media by removing the majority of existing media containing the soluble stimulant. 12 days after activation, cells were collected and further characterized.
7
Cas9-Mediated PCR-Free Oxford Nanopore Sequencing
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Genomic DNA was sheared to 20 kb fragments using Megaruptor 2 (Diagenode) and size selected using the BluePippin system (Sage Science) with a cut-off at 10 kb. 3-4 µg of sheared and size-selected DNA was prepared using the Cas9-mediated PCR-free protocol provided by Oxford Nanopore technologies with minor modifications. The crRNA and tracrRNA with Alt-R modification (Integrated DNA Technologies) were annealed in Duplex buffer (Integrated DNA Technologies) at 95°C for min and were then allowed to cool down to room temperature.
Ribonucleoproteins (RNPs) were formed by combining the annealed gRNA, HiFi Cas9 (Integrated DNA Technologies) and 1x NEB CutSmart buffer (New England Biolabs) and incubated at room temperature for 30 min. The fragmented and size-selected DNA was dephosphorylated to block all ends from ligation of adapters in a downstream adapter ligation step. Subsequently, the DNA molecules were digested by Cas9 using the previously prepared RNPs and the newly cleaved ends were dA-tailed to enable adapter ligation. The library preparation was completed by ligation of adapters from the SQK-LSK109 kit (Oxford Nanopore Technologies) and cleaned up with AMPure XP beads (Beckman Coulter) before preparation for sequencing. Sequencing was performed using the MinION system (Oxford Nanopore Technologies) with a R9.5.1 flow cell and Guppy v3.3.3 was used for base calling.
Ribonucleoproteins (RNPs) were formed by combining the annealed gRNA, HiFi Cas9 (Integrated DNA Technologies) and 1x NEB CutSmart buffer (New England Biolabs) and incubated at room temperature for 30 min. The fragmented and size-selected DNA was dephosphorylated to block all ends from ligation of adapters in a downstream adapter ligation step. Subsequently, the DNA molecules were digested by Cas9 using the previously prepared RNPs and the newly cleaved ends were dA-tailed to enable adapter ligation. The library preparation was completed by ligation of adapters from the SQK-LSK109 kit (Oxford Nanopore Technologies) and cleaned up with AMPure XP beads (Beckman Coulter) before preparation for sequencing. Sequencing was performed using the MinION system (Oxford Nanopore Technologies) with a R9.5.1 flow cell and Guppy v3.3.3 was used for base calling.
8
Tobacco PDS and ADF Gene Editing
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2.1-Plant material, resequencing of target genes and gRNA design Genomic DNA was extracted from wild-type tobacco plants (Nicotiana tabacum cv Petit Havana SR1) (3-4 weeks old, when the plants had reached the 2-3 leaf stage; Pospíšilová et al. 1998 ) using the NucleoSpin Plant II kit (Macherey-Nagel). The target regions of the selected PDS and ADF genes were ampli ed from genomic DNA using Q5 high-delity DNA polymerase (NEB). The PCR products were puri ed from agarose gels using the NucleoSpin gel and PCR clean-up kit (Macherey-Nagel) and sequenced using the Sanger method prior to gRNA design, using the primers listed in the Table S1. The gRNA sequences were designed using the Crispr RGEN Tools, Cas-Designer and CRISPR-P v2.0 online.
The gRNA targeting the PDS gene was 5′-TTTTTTTGGAATATCAGGTTTGG-3′ and the gRNA targeting the ADF gene was 5′-CTTGGAGCTGAAGAGGAAGAAGG-3′. BLAST analysis was used to identify any potential off-targets in the crRNA sequences. To prepare RNP complexes, we used crRNA, tracrRNA labeled with ATTO-550 and high delity Cas9 (HiFi Cas9) synthesized by Integrated DNA Technologies. We used crRNA XT for all experiments, which has additional chemical modi cations to optimize stability and performance.
The gRNA targeting the PDS gene was 5′-TTTTTTTGGAATATCAGGTTTGG-3′ and the gRNA targeting the ADF gene was 5′-CTTGGAGCTGAAGAGGAAGAAGG-3′. BLAST analysis was used to identify any potential off-targets in the crRNA sequences. To prepare RNP complexes, we used crRNA, tracrRNA labeled with ATTO-550 and high delity Cas9 (HiFi Cas9) synthesized by Integrated DNA Technologies. We used crRNA XT for all experiments, which has additional chemical modi cations to optimize stability and performance.
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