SpyCas9 NLS — Streptococcus pyogenes Cas9
NovoHelix manufactures high-quality Cas editors for sensitive applications such as DNA-writing and DNA-editing of early mammalian embryos. During early developmental stages such as the one-cell zygote or two-cell blastomere, microinjection or electroporation is used to deliver Cas9 as ribonucleoprotein (RNPs) complexes to introduce germline genetic modifications for generation of biomedical animal models. Each lot of NovoHelix’s SpyCas9 NLS is functionally tested in mouse zygotes by knock-in of a fluorescent reporter at the Rosa26 safe harbor locus. SpyCas9 NLS contains bipartite nuclear localization sequence (NLS) on the N- and C- termini of the protein.
Spy Cas9 NLS
Spy Cas9 NLS
Spy Cas9 NLS
Spy Cas9 NLS
Spy Cas9 NLS
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Product Literature & Protocol
FAQs & Troubleshooting
- Functional knockout of primary cells and human pluripotent cell lines
- Precise genome editing by microinjection of preimplantation mammalian embryos
- RNP electroporation of cells and zygotes
- Improved homology-directed repair (HDR) for knock-in of large DNA cassettes
- CRISPR editing detection by a mismatch nuclease to validate guide RNA activities
Spy Cas9 NLS is a class 2, Cas type II endonuclease with RuvC-like and HNH domains. The Spy Cas9 NLS was improved through protein engineering efforts. The final engineered protein was synthesized as a codon-optimized minigene, was expressed heterologously in an E. coli strain with the assistance of chaperone proteins and was purified by proprietary chromatographic techniques. This product is manufactured in the USA using chemically-defined components and, therefore, does not contain manufacturing components such as serum or animal-derived raw materials. Spy Cas9 NLS is suitable for sensitive applications such as pronuclear zygotic microinjection of Cas9 ribonucleoprotein (RNP) complexes in mouse, rat, pig, cow and goat zygotes.
>98 % by SDS-PAGE and analysis by protein mass spectrometry. The recombinant protein is free of residual chromosomal DNA from the expression chassis (E. coli strain B) as determined by a quantitative real-time PCR assay.
Purified recombinant protein was subjected to peptide mass fingerprinting (PMF) by MALDI-TOF/TOF (PMF + MS/MS).
10X Spy Cas9 NLS Reaction Buffer:
200 mM HEPES pH 6.5 @ 25°C1 M NaCl50 mM MgCl21 mM EDTA
Microinjection of mouse zygotes. The Spy Cas9 NLS endonuclease was tested for functional activity by direct microinjection of ribonucleoprotein complexes containing 400 nM Spy Cas9 NLS protein, 25 ng/ul sgRNA and 20 ng/ul supercoiled donor vector Rosa26-H2b-mCherry into pronuclear stage zygotes. Mouse ∼E13.5 C57BL/6J embryos were collected and visualized for fluorescence of mCherry, and screened by long-range junction PCRs for knock-in at the safe harbor locus Rosa26. Additional PCR assays were designed to screen across the guide RNA target site and these amplicons were submitted for Sanger sequencing to determine animals containing indel mutations. Survival rates of zygotes post-microinjection from mock injection controls and RNP injected zygotes were greater than 80% and 70% reached ~E13.5. Greater than 90% of surviving ~E13.5 embryos were modified by Spy Cas9 NLS endonuclease activity to pass lot functional testing criteria.
Slide electroporation of one-cell zygotes. To functionally test Spy Cas9 NLS gene editing activity in livestock embryos including pigs, cows and goats, diploid parthenotes were generated from abattoir-collected oocytes. Spy Cas9 NLS protein, a fluorescent 5’-ATTO-labeled tracrRNA, and Rosa26 crRNA were electroporated using a square wave pulse generator in a microscopic glass slide format containing 50 zygotic parthenotes placed in a line between the 1 mm electrode gap. Parthenotes were recovered after overnight culture, and individual embryos were lysed for whole genome amplification (WGA). Genotyping of WGA material from individual parthenotes was performed by a PCR assay that spans the Rosa26 target site. Amplicons were submitted for Sanger sequencing, and submitted to Synthego's ICE (Inference of CRISPR Edits) analysis tool. Greater than 90% of surviving parthenotes were modified by Spy Cas9 NLS endonuclease activity to pass lot functional testing criteria. Survival rates of parthenotes after slide electroporation from mock (Spy Cas9 NLS only, no RNA) controls and experimental group with Cas9 RNP complexes were greater than 90%.
Residual Nuclease Activity:
Spy Cas9 NLS is free from detectable RNase A, endonuclease (nicking) and non-specific DNase activities as determined by fluorometric assays using cleavable fluorescent-labeled substrates to be less than the limit of quantitation ≤ 0.3 pg/ml.
Lipopolysaccharide (LPS) was determined by the standard LAL (Limulus amebocyte lysate) test to be < 0.5 EU/µg. Due to potential environmental sustainability concerns (collection of the hemolymph used in pharmaceutical testing may negatively affect horseshoe crab populations), a quantitative and more sensitive endotoxin assay was developed using a pyrogen‐testing cell model with knock-in (targeted, single-copy integration) of TLR4/CD14/MD2 at the safe harbor locus AAVS1/PPP1R12C on human chromosome 19. The TLR4/CD14/MD2 assay is used as an orthogonal screen for LPS in lieu of the LAL method and has a detection limit of 0.005 EU/ml.
A sterile, aqueous, clear and colorless solution.
Spy Cas9 is supplied reconstituted at 10 mg/ml in a stabilization buffer containing 20 mM HEPES, 300 mM NaCl, 0.1 mM EDTA, 50% (v/v) glycerol at a pH ~7.5. The reconstituted solution has been filter sterilized by passing through a 0.22 micron PES membrane and tested to be negative for mycoplasma contamination.
Spy Cas9 NLS is supplied at 10 mg/ml in stabilization buffer and remains bioactive for 10 days if maintained refrigerated 2º to 8ºC (35º to 46ºF). Stock solutions of Spy Cas9 NLS in its concentrated form can be stored at least 6 months at -20°C and at least 12 months at -80°C from the date of manufacture with no loss of activity. For long term storage, add 0.1% w/v carrier protein such as recombinant human serum albumin (HSA) and avoid repeated freeze-thaw cycles by aliquoting. Multiple freeze-thaw cycles reduce potency and, therefore, aliquoting working stocks is strongly recommended. To maximize recovery, spin the microcentrifuge tube prior to opening but do not vortex. For genome editing experiments such as preparation before microinjection of zygotes, prepare an RNase-free work area using only RNase-free reagents and consumables such as microcentrifuge tubes, and barrier pipette tips while setting up these experiments.
Disclaimer & Precautions:
This product is solely for research and development use only and may be subject to conditional use and licensing restrictions. The product shall not be used as an advanced pharmaceutical intermediate (API) or investigational drug or a biologic. This product is not intended to be used as a therapeutic agent or facilitate clinical diagnosis or be used as an in vitro diagnostic (IVD) product.
The Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER) define an IVD as:
“In vitro diagnostic products are those reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended for use in the collection, preparation, and examination of specimens taken from the human body. These products are devices as defined in section 201(h)of the Federal Food, Drug, and Cosmetic Act (the act), and may also be biological products subject to section 351 of the Public Health Service Act. Title 21, Code of Federal Regulations (CFR), section 809.3(a).”
This product shall not be used or formulated in any agricultural, pesticidal, veterinary or animal products, food additives or household chemicals or any other unspecified use. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. NovoHelix distributes products for basic and translational research use only. NovoHelix will report any unspecified use to respective regulatory authorities for enforcement to ensure safeguarding of our research products from potential abuse.
Notice to purchaser:
The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents or applications owned by NovoHelix to use this product. No other license under these patents or applications is conveyed expressly or by implication by purchase of this product.
Product Literature & Protocol
- HiFi DNA Assembly Protocol
Recommended Amount of Fragments Used for Assembly
|2–3 Fragment Assembly*||4–6 Fragment Assembly**||Positive Control✝|
|Recommended DNA Molar Ratio||vector:insert = 1:2||vector:insert = 1:1|
|Total Amount of Fragments|
HiFi DNA Assembly Master Mix
|10 μl||10 μl||10 μl|
|Deionized H2O||10-X μl||10-X μl||0|
|Total Volume||20 μl✝✝||20 μl✝✝||20 μl|
* Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.
**To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).
† Control reagents are provided for 5 experiments.
†† If greater numbers of fragments are assembled, increase the volume of the reaction linearly by using additional NovoHelix HiFi DNA Assembly Master Mix. Alternatively, pool the DNA fragments into an equimolar mix first and then re-purify these pooled equimolar fragments over a micro-column and elute with a minimum volume (~10-µl). The eluate may be reapplied to the same micro-column membrane to improve elution of large DNA fragments without increasing the final volume..
Recommended Storage Condition:
This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.
FAQs & Troubleshooting
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