The CRISPR RNP was delivered into cells by nucleofection. For one nucleofection, 6 µg SpCas9 protein (Cat# 1081059, IDT) and the 3.2 µg synthetic gRNA (Synthego) was mixed in a PCR tube by pipetting and incubated at room temperature for at least 10 min and maximum 1 h. Then 200,000 suspended cells were gently resuspended cells in 20 uL nucleofection buffer (OptiMEM) by pipetting up and down. The cells and RNP complex were then transferred to a 4D-Nucleofector 16-well nucleocuvette strip (Catalog #: AXP-1004, Lonza). The samples should cover the bottom of the wells, and any presence of air bubble must be avoided. Nucleofection was performed with program CM-138. Immediately after electroporation, prewarmed culture media was added to the cells (180 µL per well of the Nucleocuvette strip). The cells were then transferred into one well of a 12-well cell culture plate with prewarmed medium. 48 h after transfection, cells were harvested for amplicon PCR and deep sequencing.
Synthetic grna
Manufactured by Synthego
Sourced in United States
Synthego's Synthetic gRNA is a laboratory product designed for use in gene editing applications. It serves as a crucial component in the CRISPR-Cas9 system, providing the guide RNA sequence that directs the Cas9 enzyme to the target DNA sequence. The Synthetic gRNA is engineered to be highly specific and effective in gene editing experiments.
Automatically generated - may contain errors
Lab products found in correlation
4d nucleofector, by Lonza (1 mentions)
Immunocult human cd3 cd28 cd2 t cell activator, by STEMCELL (1 mentions)
Spcas9 protein, by PNA Bio (1 mentions)
Kit p3, by Lonza (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Lipofectamine messengermax, by Thermo Fisher Scientific (1 mentions)
Optimem serum reduced medium, by Thermo Fisher Scientific (1 mentions)
Cas9 nls protein, by PNA Bio (1 mentions)
5 protocols using synthetic grna
1
CRISPR RNP Delivery by Nucleofection
2
CRISPR Editing of Activated PBMCs
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PBMCs were thawed and activated using ImmunoCult Human CD3/CD28/CD2 T-cell Activator (5 µl/106 cells) (STEMCELL Technologies, Vancouver, Canada, cat# 10970). Three days later, ribonucleoprotein (RNP) complexes were transferred to 1 × 106 cells using the EO-115 program (4D-Nucleofector, Lonza, Basel, Switzerland) and kit P3 (Lonza, cat# V4XP-3032) following the manufacturer’s instructions. Prior to electroporation, 20 pmol of SpCas9 protein (PNA Bio, Newbury Park, California, USA, cat# CP02) and 112.5 pmol of synthetic gRNA (Synthego, Silicon Valley, California, USA) were complexed by incubation for 10 min at RT. Upon electroporation, fresh complete medium with 1000 U/ml of IL-2 was added to recover the cells. The cells were split in four wells (0.25 × 106 cells/well) of a 96-well U-shaped-bottom microplate (Falcon, Fisher Scientific, Hampton, New Hampshire, USA, cat# 08-772-54) and transduced with indicated amounts of AAV vector particles 15 min post electroporation.
3
CRISPR mRNA Transfection in Primary Human Hepatocytes
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PHH cocultures were transfected 48 h after coculture generation with 3T3-J2 murine fibroblasts. Transfections with mRNA were performed using Lipofectamine MessengerMax (Thermo Fisher Scientific, LMRNA003) in accordance with manufacturers’ protocols, with the following optimized specifics: 1 µg (for saturating conditions) of mRNA encoding for editor and 333 ng of synthetic gRNA (Synthego) were combined in 30 µl of OptiMEM serum reduced medium (Gibco, 31985). A 30 µl 1:15 (Lipofectamine:OptiMEM) mixture was added to the mRNA/gRNA solution with the resulting final mixture left to rest at ambient temperature for 15 min. The entire 60-µl solution was used to treat a well of cocultured primary human hepatocytes. Each study condition was run in triplicate and transfection amounts used were scaled up accordingly. At 9 d post-transfection, the PHH cocultures were lysed with a solution of 10 mM Tris–HCl pH8.0 (Thermo Fisher Scientific, 15568025), 0.05% SDS (Thermo Fisher Scientific, 15553027) and 500 µg proteinase K (Thermo Fisher Scientific, EO0491) at a total of 200 µl per well. Once lysed, lysate was treated at 85 °C for 15 min to inactivate proteinase K. The sequences of sgRNAs used in this study are specified in Supplementary Table 3 .
4
CRISPR-Mediated Gene Editing in Embryos
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Coding sequences were selected for CRISPR targeting based on whether they are conserved in both the S and L gene variants, encode conserved protein domains required for protein function, and are considered optimized for CRISPR mutagenesis (https://chopchop.cbu.uib.no ; table S6). Sixty picomoles of a synthetic gRNA (Synthego) was mixed with 12 pmol of NLS-Cas9 protein (PNA Bio) in a total volume of 5 μl and one injection of 20 nl was made into the animal pole of embryos, between 25 and 40 min after fertilization. Compound mutants were injected successively with different gRNA/Cas9 mixtures. At the two- to four-cell stage, embryos were injected with experimental or tracer RNAs.
CRISPR targeting of genes and the specificity of the mutant phenotypes were assessed in this study using different approaches. In some cases (emi2 and plk1), at least two gRNAs were tested to control for offsite mutations. In addition, efficient editing and indel production in embryos injected with Cas9/emi2gRNA1 was assessed using the RNA-seq analysis, using reads that mapped to the L and S forms of the emi2 gene (fig. S1K). Last, in some cases, we assess protein levels (plk1) since F0 crispants potentially leave maternal RNA intact, which can potentially mask mutant phenotypes, especially in cases where the gene is not up-regulated during MCC differentiation.
CRISPR targeting of genes and the specificity of the mutant phenotypes were assessed in this study using different approaches. In some cases (emi2 and plk1), at least two gRNAs were tested to control for offsite mutations. In addition, efficient editing and indel production in embryos injected with Cas9/emi2gRNA1 was assessed using the RNA-seq analysis, using reads that mapped to the L and S forms of the emi2 gene (fig. S1K). Last, in some cases, we assess protein levels (plk1) since F0 crispants potentially leave maternal RNA intact, which can potentially mask mutant phenotypes, especially in cases where the gene is not up-regulated during MCC differentiation.
5
Generating Isogenic iPSC-CMs with TTN Variants
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