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Protocols for CRISPR genome editing in your model system

Tips for using Alt-R® CRISPR Systems—send us yours!

Research into applications using CRISPR-Cas systems for genome editing is expanding quickly. This presents a need for effective reagents and protocols for a wide variety of model systems beyond mammalian cultured cells.

Advantages of Alt-R CRISPR RNAs and proteins for genome editing

CRISPR guide RNAs. Use Alt-R CRISPR RNAs to direct potent, on-target genome editing. These length-optimized RNAs are chemically synthesized, which allows addition of modifications for increased nuclease resistance and reduced innate immune responses.

CRISPR proteins. Choose from several recombinant variants of Cas9 (Streptococcus pyogenes), as well as Cpf1 (Acidaminococcus sp. BV3L6) endonucleases. Alt-R S.p. Cas9 Nuclease 3NLS is suitable for most genome editing studies. Some experiments may benefit from use of Alt-R S.p. HiFi Cas9 Nuclease, a high-fidelity nuclease that has been engineered to reduce off-target effects while retaining the on-target potency of wild-type Cas9. Alt-R A.s. Cpf1 Nuclease 2NLS provides additional genome editing target sites, because it recognizes an AT-rich protospacer adjacent motif (PAM) rather than the CG-rich Cas9 PAM sequence.

If you are interested in knock-in experiments or other applications requiring homology-directed repair (HDR), consider using an Alt-R Cas9 nickase to create single-strand breaks. Use of a nickase variant with a pair of crRNAs reduces off-target effects by requiring specific binding of 2 ribonucleoproteins (RNPs), and promotes homology-directed repair, in part, by creating overhanging DNA breaks. However, you will need 2 potent CRISPR target sites at an appropriate distance from your HDR target site.

Alt-R CRISPR RNPs. While the Alt-R CRISPR RNAs are compatible with CRISPR proteins from any source (e.g., cells that stably express CRISPR protein, or CRISPR proteins expressed from DNA or mRNA constructs), we recommend delivery of CRISPR reagents as RNPs by lipofection (e.g., Figure 1), electroporation, or microinjection. Cas9 RNPs are compatible with all 3 methods; however, for Cpf1 RNPs, we recommend electroporation and microinjection delivery methods.

Advantages of using Alt-R RNPs include:

  • Efficient, on-target genome editing
  • Precise control of editing complex delivery—cells will not contain templates that express unregulated levels of CRISPR RNAs or endonucleases
  • Lower activation of cellular immune responses and, therefore, reduced toxicity—toxicity is often observed when using in vitro transcribed Cas9 mRNA or single guide RNAs (sgRNA)

 

Figure 1. Lipofection of Alt-R CRISPR-Cas9 system components as a ribonucleoprotein (RNP) outperforms other transient CRISPR-Cas9 methods. Alt-R CRISPR HPRT Control crRNA for human, mouse, or rat were complexed with Alt-R CRISPR tracrRNA. Resulting complexes were transfected with Cas9 expression plasmid, Cas9 mRNA, or as part of a Cas9 RNP (containing Alt-R S.p. Cas9 Nuclease 3NLS, pre-complexed with the crRNA and tracrRNA) into human (HEK-293), mouse (Hepa1-6), or rat (RG2) cell lines. The Cas9 RNP outperformed the other transient Cas9 expression methods, and performed similar to reference HEK293-Cas9 cells that stably express S. pyogenes Cas9.

Protocols and starting points are increasingly available

IDT scientists have developed detailed lipofection and electroporation protocols for using the Alt-R CRISPR-Cas9 System and the Alt-R CRISPR-Cpf1 System in mammalian cells (Table 1). With help from our collaborators, we also make user methods available as starting points for genome editing in other model systems. These protocols and methods are a good starting point for protocol optimization for your studies, and often offer tips for all aspects of genome editing experiments, from growth conditions through genome editing detection.

Table 1. Current Alt-R CRISPR System protocols and user methods.*

* Visit www.idtdna.com/protocols to access the most up-to-date list of protocols and user-submitted methods. Visit the Alt-R CRISPR-Cas9 citations page to find peer-reviewed publications describing research using the Alt-R CRISPR-Cas9 system in yeast, primary human T cells, and more.
Model system Transfection method IDT protocols
Cultured cells Lipofection [1]
Cultured cells Electroporation [2–6]
 In vitro— [7]  
Model system Transfection method User methods
Human pluripotent or embryonic stem cells Electroporation [8]
Mouse induced pluripotent stem cells Electroporation [9]
Mouse zygote Electroporation [10,11]
Mouse zygote Microinjection [12]
Zebrafish embryo Microinjection [13]
C. elegans Injection [14]

Do you have a protocol to share?

We are always interested in learning about successful editing in different model systems, and would like to serve as your resource for information about genome editing applications. If you have a novel protocol for using Alt-R CRISPR RNA and/or nucleases that you would like to share with the research community, email us at CRISPR@idtdna.com to start the discussion.

References

  1. Integrated DNA Technologies. (2017) Alt-R CRISPR-Cas9 System: Cationic lipid delivery of CRISPR ribonucleoprotein complex into mammalian cells. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  2. Integrated DNA Technologies. (2017) Alt-R CRISPR-Cas9 System: Delivery of ribonucleoprotein complexes in HEK-293 cells using the Amaxa® Nucleofector® System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  3. Integrated DNA Technologies. (2017) Alt-R CRISPR-Cas9 System: Delivery of ribonucleoprotein complexes in HEK-293 cells using the Neon® Transfection System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  4. Integrated DNA Technologies. (2017) Alt-R CRISPR-Cas9 System: Delivery of ribonucleoprotein complexes into Jurkat T cells using the Bio-Rad Gene Pulser® Xcell™ Electroporation System. [Online] Coralville, Integrated DNA Technologies. (Accessed December 21, 2017.)
  5. Integrated DNA Technologies. (2017) Alt-R CRISPR-Cpf1 System: Delivery of ribonucleoprotein complexes in HEK-293 cells using the Amaxa Nucleofector System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  6. Integrated DNA Technologies. (2017) Alt-R CRISPR-Cpf1 System: Delivery of ribonucleoprotein complexes in Jurkat T cells using the Neon Transfection System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  7. Integrated DNA Technologies. (2017) Alt-R CRISPR-Cas9 System: In vitro cleavage of target DNA with ribonucleoprotein complex. [Online] Coralville, Integrated DNA Technologies. (Accessed December 21, 2017.)
  8. Moore K, Martin J. (2017) Electroporation of human pluripotent or embryonic stem cells with CRISPR reagents. [Online] Coralville, Integrated DNA Technologies. (Accessed December 21, 2017.)
  9. Nepa Gene. (2017) Electroporation of human induced pluripotent stem (iPS) cells. [Online] Coralville, Integrated DNA Technologies. (Accessed December 21, 2017.)
  10. Hashimoto M, Takemoto T. (2017) Mouse zygote electroporation: Ribonucleoprotein delivery using the Alt-R CRISPR-Cas9 System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  11. Nepa Gene. (2017) Mouse zygote electroporation: RNP delivery using teh Alt-R CRISPR-Cas9 System and the NepaGene21 Electroporator.  [Online] Coralville, Integrated DNA Technologies. (Accessed December 21, 2017.)
  12. Quadros R, Harms D, Gurumurthy CB. (2016) Mouse zygote microinjection: Ribonucleoprotein delivery using the Alt-R CRISPR-Cas9 System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  13. Essner J. (2016) Zebrafish embryo microinjection: Ribonucleoprotein delivery using the Alt-R CRISPR-Cas9 System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)
  14. Kohler S, Dernburg A. (2016) C. elegans injection: Ribonucleoprotein delivery using the Alt-R CRISPR-Cas9 System. [Online] Coralville, Integrated DNA Technologies. (Accessed October 13, 2017.)

Product focus—genome editing tools

Alt-R CRISPR Systems—all of the reagents needed for successful genome editing.

  • Achieve higher efficiency genome editing than other methods by using Alt‑R CRISPR RNAs, which are optimized, nuclease resistant, and individually quality controlled
  • Avoid toxicity or innate immune response activation by using Alt-R CRISPR ribonucleoproteins, instead of in vitro transcribed CRISPR nuclease mRNA and sgRNAs
  • Select from 2 CRISPR systems based on Cas9 or Cpf1 endonuclease for increased target site options
  • Extend the ease of use and performance of the Alt-R system with additional CRISPR reagents for enhanced RNP electroporation, fluorescent visualization, and genome editing detection.

Learn more at www.idtdna.com/CRISPR.


Ultramer® Oligonucleotides—are high-fidelity, single-stranded DNA sequences up to 200 nt. Have them custom synthesized to contain your insertion sequence and homology arms for use in CRISPR HDR experiments.They are also useful in applications such as gene synthesis and mutagenesis, and as qPCR standards.

Learn more at www.idtdna.com/Ultramer.


gBlocks® Gene Fragments—these double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, shipped in 2–5 working days, are perfect for quick, affordable, and easy gene construction or modification. The dsDNA fragments have been used in CRISPR-mediated genome editing, as well as a wide range of other applications, including antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.

Learn more at www.idtdna.com/gBlocks.


Additional resources

Alt-R CRISPR-Cas9 System

Applications of Cas9 nickases for genome editing—Application note: Learn tips for designing guide RNA pairs for optimized genome editing and donor templates for homology-directed repair.

Use of fluorescently labeled Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550—Application note: Learn how fluorescently labeled tracrRNA can be used to detect and visualize cells that have been successfully transfected with an RNP.

Visit the Alt-R CRISPR-Cas9 citations page to find peer-reviewed publications describing research using the Alt-R CRISPR-Cas9 system.


Alt-R CRISPR-Cpf1 System

Cpf1 genome editing using the Alt-R CRISPR-Cpf1 System—Webinar: Watch a recording of our webinar to learn about the components of the Alt-R CRISPR-Cpf1 System, as well as the strengths and weaknesses of this powerful genome editing research tool.


Author: Maureen Young, PhD, is a senior scientific writer at IDT.


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