Log In Sign up
The largest database of trusted experimental protocols

Spcas9 protein

Manufactured by Aldevron
Visit Supplier

SpCas9 protein is a highly purified recombinant Streptococcus pyogenes Cas9 protein. It is a key component in CRISPR-Cas9 gene editing technology, responsible for the DNA-cleaving activity.

Automatically generated - may contain errors

Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Pcag cre gfp, by Addgene (2 mentions) Accutase, by Merck Group (2 mentions) Puromycin, by Merck Group (2 mentions) Puromycin, by Addgene (2 mentions) Stop solution, by BioLegend (1 mentions) Electroporation enhancer, by Integrated DNA Technologies (1 mentions) P3 buffer, by Lonza (1 mentions) Il 7 and il 15, by Miltenyi Biotec (1 mentions) Texmacs medium, by Miltenyi Biotec (1 mentions)

5 protocols using spcas9 protein

1

Generation of LRRK2 Knock-in iPS Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
A million of LRRK2 or 100550A iPS cells were harvested using Accutase (Sigma) and reverse-transfected with 1 μg of donor construct, 12 pmol spCas9 protein (Aldevron), and 18 pmol of sgRNA (LRRK2: 5′-GAACTCACATCTGAGGTCAG-3′, AARS1: 5′-GGGCGTATCGGACAGCTCGG-3′, Synthego), 4 μl P3000 reagent and later with 5 μl Lipofectamine 3000 (Thermo-Fisher). A mixture of transfection reagents was added onto a Cultrex-coated well first and then followed by resuspended LRRK2 iPS cells or 100550A iPS cells in fresh medium with 5 μM Y-27632 (Stemgent). Puromycin (500 ng/ml, Sigma) was added into the medium three to five days after transfection. Drug-resistant cells were replated at low density (5,000 cells/100 mm dish) and single cell colonies were manually selected afterwards. Clones with both 5’ and 3’ insertion positive genotyping results were further expanded and the Puromycin cassette was deleted by transient transfection of a CRE vector pCAG-Cre:GFP (Addgene #13776) and then plated at low density for single cell colonies. After a 2nd round of genotyping, positive clones were expanded and characterized.
Cai J., Kropf E., Hou Y.M, & Iacovitti L. (2021). A stress-free strategy to correct point mutations in patient iPS cells. Stem cell research, 53, 102332.
+ Open protocol
+ Expand
2

Quantifying Anti-Cas9 Antibody Response

Check if the same lab product or an alternative is used in the 5 most similar protocols
The anti-Cas9 antibody response was measured using ELISA. Briefly, a 96-well plate was coated with 0.1 μg per well of SpCas9 protein (Aldevron, 9212) for 3 hours at 37°C. Subsequently, the plate was washed four times and then blocked by adding 100 μl of assay diluent buffer (BioLegend, 421203) for 1 hour at 25°C. Sera from mice injected with LNP-CRISPR-mAT or AAV9-Cas9 were diluted 40 times, and the plate was incubated with the samples for 2 hours at 25°C. After washing, the plate was incubated with anti-mouse IgG–conjugated with horseradish peroxidase (HRP; Sigma, A9044; Sigma-Aldrich), subsequently washed, and then treated with trimethylboron (TMB) substrate (BioLegend, 421101). After stopping the HRP/TMB reaction using stop solution (BioLegend, 423001), the plate was subjected to absorbance measurement at 450 nm. Commercial mouse anti-Cas9 antibody (Abcam, 191468) was used for a standard curve test, and the standard curve (R2 = 0.989) was used for determining anti-Cas9 IgG concentrations of the tested samples.
Han J.P., Kim M., Choi B.S., Lee J.H., Lee G.S., Jeong M., Lee Y., Kim E.A., Oh H.K., Go N., Lee H., Lee K.J., Kim U.G., Lee J.Y., Kim S., Chang J., Lee H., Song D.W, & Yeom S.C. (2022). In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy. Science Advances, 8(3), eabj6901.
+ Open protocol
+ Expand
3

Generation of LRRK2 Knock-in iPS Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
A million of LRRK2 or 100550A iPS cells were harvested using Accutase (Sigma) and reverse-transfected with 1 μg of donor construct, 12 pmol spCas9 protein (Aldevron), and 18 pmol of sgRNA (LRRK2: 5′-GAACTCACATCTGAGGTCAG-3′, AARS1: 5′-GGGCGTATCGGACAGCTCGG-3′, Synthego), 4 μl P3000 reagent and later with 5 μl Lipofectamine 3000 (Thermo-Fisher). A mixture of transfection reagents was added onto a Cultrex-coated well first and then followed by resuspended LRRK2 iPS cells or 100550A iPS cells in fresh medium with 5 μM Y-27632 (Stemgent). Puromycin (500 ng/ml, Sigma) was added into the medium three to five days after transfection. Drug-resistant cells were replated at low density (5,000 cells/100 mm dish) and single cell colonies were manually selected afterwards. Clones with both 5’ and 3’ insertion positive genotyping results were further expanded and the Puromycin cassette was deleted by transient transfection of a CRE vector pCAG-Cre:GFP (Addgene #13776) and then plated at low density for single cell colonies. After a 2nd round of genotyping, positive clones were expanded and characterized.
Cai J., Kropf E., Hou Y.M, & Iacovitti L. (2021). A stress-free strategy to correct point mutations in patient iPS cells. Stem cell research, 53, 102332.
+ Open protocol
+ Expand
4

Generation of HDR Donor Templates for AAV6 and IDLV

Check if the same lab product or an alternative is used in the 5 most similar protocols
AAV6 donor templates for HDR were generated from a construct containing AAV2 inverted terminal repeats, produced by TIGEM Vector Core by triple‐transfection method and purified by ultracentrifugation on a cesium chloride gradient. IDLV donor templates for HDR were produced exploiting HIV‐derived, third‐generation self‐inactivating transfer constructs. IDLV stocks were prepared and titered as previously described (Lombardo et al, 2007). dsDNA donor templates for HDR were obtained by restriction digestions or synthetized by high‐fidelity PCR. dsDNA digestions or amplicons were then purified by gel electrophoresis in order to remove plasmid contamination. Primers for linearization are listed in Appendix Table S5. Schematic designs of each donor construct with homologies for the CD40LG gene are reported throughout the paper.
Ribonucleoproteins (RNPs) were assembled by incubating at 1:2 molar ratio either S.p.Cas9 protein (Aldevron) or Hi‐Fi Cas9 protein (where indicated; Aldevron) with synthetic cr:tracrRNA (Integrated DNA Technologies) for 10′ at 25°C. Electroporation enhancer (Integrated DNA Technologies) was added prior to electroporation according to manufacturer’s instructions, only when treating CD34+ cells.
Vavassori V., Mercuri E., Marcovecchio G.E., Castiello M.C., Schiroli G., Albano L., Margulies C., Buquicchio F., Fontana E., Beretta S., Merelli I., Cappelleri A., Rancoita P.M., Lougaris V., Plebani A., Kanariou M., Lankester A., Ferrua F., Scanziani E., Cotta‐Ramusino C., Villa A., Naldini L, & Genovese P. (2021). Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper‐IgM syndrome. EMBO Molecular Medicine, 13(3), e13545.
+ Open protocol
+ Expand
5

CRISPR-Cas9 Mediated Gene Editing of T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
CD4 and CD8 T cells were positively enriched from healthy donor leukapheresis products by magnetic selection (Miltenyi) and activated for 48 h with CD3 and CD28 stimulation (TransACT, 1:17.5 by volume; Miltenyi) in T cell medium (TexMACS medium, Miltenyi, supplemented with 12.5 ng ml–1 IL-7 and IL-15, Miltenyi, and 3% human AB serum, Valley Biomedical). After activation, the T cells were centrifuged and resuspended in P3 buffer (Lonza). CRISPR–Cas ribonucleoproteins (RNPs) were formulated by complexing guide RNAs targeting TRAC and TRBC (Synthego) to spCas9 protein (Aldevron) in a 6:1 molar ratio. The patient-specific HR template and RNPs were mixed with the cell suspension, electroporated (Lonza, X-unit, EO-115) and transferred into T cell medium in a 24-well G-rex (Wilson Wolf) for 4–5 days with changes of the medium as needed.
Foy S.P., Jacoby K., Bota D.A., Hunter T., Pan Z., Stawiski E., Ma Y., Lu W., Peng S., Wang C.L., Yuen B., Dalmas O., Heeringa K., Sennino B., Conroy A., Bethune M.T., Mende I., White W., Kukreja M., Gunturu S., Humphrey E., Hussaini A., An D., Litterman A.J., Quach B.B., Ng A.H., Lu Y., Smith C., Campbell K.M., Anaya D., Skrdlant L., Huang E.Y., Mendoza V., Mathur J., Dengler L., Purandare B., Moot R., Yi M.C., Funke R., Sibley A., Stallings-Schmitt T., Oh D.Y., Chmielowski B., Abedi M., Yuan Y., Sosman J.A., Lee S.M., Schoenfeld A.J., Baltimore D., Heath J.R., Franzusoff A., Ribas A., Rao A.V, & Mandl S.J. (2022). Non-viral precision T cell receptor replacement for personalized cell therapy. Nature, 615(7953), 687-696.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!