Ps2Ex3 sgRNA (90 ng/μL final concentration) was mixed with Cas9 nuclease (Invitrogen, Carlsbad, California, USA, B25640), and then incubated at 37°C for 15 min to maximize cleavage efficiency after injection. Tübingen strain wild type zebrafish embryos were generated by mass mating and injected at the one-cell stage with 5–10 nL of this mixture. The injected embryos were subsequently raised for mutation screening.
Cas9 nuclease
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cas9 nuclease is a protein-based gene-editing tool derived from the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. It functions as a molecular scissors, capable of precisely cutting targeted DNA sequences. Cas9 is a key component in the CRISPR-Cas9 system, enabling the modification of specific genetic information.
Automatically generated - may contain errors
Lab products found in correlation
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
Geneart crispr grna design tool, by Thermo Fisher Scientific (2 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (2 mentions)
Ribomax large scale rna production system, by Promega (1 mentions)
Neon electroporation, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
11 protocols using cas9 nuclease
1
Targeted Zebrafish Mutagenesis via CRISPR
2
Efficient CRISPR-Cas9 Genome Editing in iPSCs
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DNA oligonucleotides used for gRNA targeting were designed with the GeneArt CRISPR gRNA Design Tool (Thermo Fisher Scientific; Invitrogen Life Technologies, Carlsbad, CA, USA). To examine the cleavage efficiency of gRNA, a series of gRNAs flanking the target site was designed and synthesized. Each individual gRNA was combined with Cas9 nuclease (Invitrogen Life Technologies, Carlsbad, CA, USA) to form Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs). The Cas9 RNPs were then used to transfect the iPSCs, a task for which the Neon Transfection System (Invitrogen Life Technologies, Carlsbad, CA, USA) was used. Genomic editing efficiency was then evaluated through T7 Endonuclease I (T7E1) assay 48 h after transfection. The gRNAs that had both the highest cleavage efficiencies and closest proximity to the target site were selected for the subsequent genome editing. For precise genome editing, the Cas9 RNPs and repair template (ssODN from IDT, Coralville, IA, USA) were coelectroporated into the iPSCs. The transfected iPSCs were then clonally expanded to derive isogenic cell lines. The single-nucleotide substitution was screened using a TaqMan SNP Genotyping Assay (Applied Biosystems, Forster City, CA, USA) and confirmed through Sanger sequencing.
3
Efficient CRISPR-Cas9 Mediated Mutagenesis
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Tübingen (TU) strain wild type embryos were collected from mass spawning. The target sgRNA (70 ng/μL final concentration) was mixed with “N140I oligo” (30 ng/μL for final concentration) and Cas9 nuclease (1μg/μL for final concentration) (Invitrogen, Carlsbad, California, USA, B25640), and then incubated at 37°C for 15 min to maximize formation of active CRISPR Cas9 complexes. 5–10 nL of the mixture was then injected into zebrafish embryos at the one-cell stage. The injected embryos were subsequently raised for mutation screening.
4
CRISPR Guide RNA Design and Production
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CRISPRdirect (http://crispr.dbcls.jp/doc/ ) online software was used to screen appropriate single-guide RNA (sgRNA) target sequences. The gRNAs were synthetized by the Beijing Genomics Institute. The sgRNA templates were transcribed using T7 polymerase with RiboMAX Large-Scale RNA Production Systems (Promega, Beijing, China) according to the manufacturer’s instructions. The RNA transcripts were purified using 3 M sodium acetate (pH 5.2) and anhydrous ethanol (1:30) precipitation, washed with 75% ethanol, and eluted in nuclease-free water. All injection mixtures contained 300 ng/µl Cas9 nuclease (Invitrogen, California, USA) and 300 ng/µl purified sgRNA. Before injection, mixtures of Cas9 nuclease and gRNA were incubated for 15 min at 37°C to reconstitute active RNPs (Zou et al., 2022 (link)).
5
Generating Genetically Modified Monkey Embryos
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Mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) immediately and then cultured in CMRL-1066 containing 10% fetal bovine serum (FBS; Invitrogen, 16140071) at 37°C in 5% CO2. Fertilization was confirmed by the presence of a second polar body and two pronuclei. To produce BRN2-knockout embryos, we injected a mixture of sgRNA pair (2500 ng/μl, 1250 ng/μl for each sgRNA) and Cas9 nuclease (5000 ng/μl; Invitrogen, A36499) with a total volume of 5 pl into each oocyte before ICSI. Microinjections were performed in the cytoplasm of oocytes using a microinjection system under standard conditions. Zygotes (wild-type and BRN2 RNP-treated) were then cultured in chemically defined hamster embryo culture medium-9 (HECM-9) containing 10% FBS (Gibco, 16140071) at 37°C in 5% CO2 to allow embryo development. The culture medium was replaced every other day until the blastocyst stage. The cleaved embryos with high quality at the two-cell to blastocyst stage were transferred into the oviduct of the matched recipient mother monkeys. Thirty-eight monkeys were used as surrogate recipients. The earliest pregnancy diagnosis was performed by ultrasonography about 20 days after the embryo transfer. Both clinical pregnancy and number of fetuses were confirmed by fetal cardiac activity and the presence of a yolk sac as detected by ultrasonography.
6
CRISPR-Cas9 for 18S Amplicon Cleavage
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For each of the ten host organisms and the mock community of protists and fungi, the purified 18S amplicons were cut using Cas9 nuclease, S. pyogenes (NEB) in the presence of a sgRNA, following the manufacturer’s directions. Briefly, the 10 μL reaction contained approximately 0.1 pmol of dsDNA, 1 pmol of sgRNA, and 1 pmol of Cas9, as well as 1x Cas9 reaction buffer to keep the molar ratio of Cas9:sgRNA:template DNA at 10:10:1. The reaction was incubated at 37 °C for 4 h in a thermocycler, followed by 70 °C for 10 min to deactivate the CRISPR-Cas9. For each sample, in parallel with the CRISPR-Cas9 treatment, we also prepared the reaction without CRISPR-Cas9 treatment, in which Cas9 nuclease and sgRNA were replaced with molecular grade ultrapure water (Invitrogen). Thus, each reaction of both treatments contained the same amount of template dsDNA (18S amplicons at 0.1 pmol) and was subjected to the same incubation conditions.
7
CRISPR-Cas9 Mediated Precise Genome Editing
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The DNA oligonucleotides used for the gRNA targeting were designed using the GeneArt CRISPR gRNA Design Tool (Thermo Fisher Scientific). To examine the cleavage efficiency of gRNA, a series of gRNAs flanking the target site were designed and synthesized. Each individual gRNA was combined with CRISPR associated protein 9 (Cas9) nuclease (Invitrogen) to form Cas9 protein/gRNA ribonucleoprotein complex (Cas9 RNP). The Cas9 RNP was then used to transfect hiPSCs via Neon Transfection System (Invitrogen). The genomic editing efficiency was evaluated by T7 endonuclease I (T7E1) assay at 48 hr post transfection. The gRNAs with highest cleavage efficiencies, and that were also in close proximity to the target site, were selected for the subsequent genome editing. For precise genome editing, the Cas9 RNPs and repair template (ssODN from IDT, Coralville, USA) were co-electroporated into hiPSCs. The transfected hiPSCs were then clonally expanded to derive isogenic cell lines. Each single-nucleotide substitution was screened using a TaqMan SNP genotyping assay (Applied Biosystems, USA) and further confirmed by Sanger sequencing.
8
Zebrafish Genome Editing via Cas9
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Tübingen (wild type embryos were generated by mass mating. Ps2Ex3 sgRNA (90 ng/μL final concentration) was first mixed with Cas9 nuclease (Invitrogen, Carlsbad, California, USA, B25640), and then incubated at 37°C for 15 min to maximize cleavage efficiency. 5-10 nL of the mixture was injected into zebrafish . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint this version posted April 20, 2020. ; https://doi.org/10.1101/2020.04.20.050815 doi: bioRxiv preprint embryos at the one-cell stage. The injected embryos were subsequently raised for mutation screening.
The copyright holder for this preprint this version posted April 20, 2020. ; https://doi.org/10.1101/2020.04.20.050815 doi: bioRxiv preprint embryos at the one-cell stage. The injected embryos were subsequently raised for mutation screening.
9
CRISPR Targeting of Key Immune Regulators
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CRISPR RNAs (crRNAs) targeting the CCR5, PRDM1, IRF4, PAX5, and BACH2 loci (sequences in Table S1 ) were identified using the MIT CRISPR design tool (http://crispr.mit.edu/ ) and the Broad Institute single guide RNA (sgRNA) design tool (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design ) and synthesized (IDT) containing phosphorothioate linkages and 2′O-methyl modifications.26 (link) ssODNs were commercially synthesized by IDT (Ultramer DNA Oligonucleotides) with phosphorothioate linkages. crRNA and trans-activating crRNA (tracrRNA; IDT) hybrids were mixed with Cas9 nuclease (IDT) at a 1.2:1 ratio and delivered with or without ssODNs to cells by Neon electroporation (Thermo Fisher Scientific).
10
CRISPR-Mediated Genetic Manipulation of HSCs
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