TechVault
  • How do I choose which two guide RNAs to multiplex, when using the Alt-R® nickases?
    There are multiple factors to consider when selecting the guide RNAs to multiplex with Cas9 nickase:
    • Each crRNA should function efficiently in individual genome editing experiments.
    • The two crRNAs must target opposite strands to ensure a double-strand break is created.
    • Distance between cleavage sites is important for optimal performance.
      • Alt-R Cas9 D10A Nickase 3NLS: 40–70 bp
      • Alt-R Cas9 H840A Nickase 3NLS: 50–70 bp
    • Outward PAM site orientation (right side)
    • Pam site orientation (Cas9 nickase experiments)

    For more information about the design and use of Cas9 nickases, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Support section).
  • When using the Alt-R® nickases, what orientation should my guides have? Should they target the same or opposite strands?

    We have found that having the PAM sites oriented to the outside of the target region (Figure) provides the most robust results. To induce a staggered double-strand break at a target location, the two guide RNAs must target opposite strands.

    PAM-out orientation (right side)
    (light blue = genomic DNA, dark blue = protospacer, and orange = PAM sequence):

    Pam site orientation (Cas9 nickase experiments)

    In addition, for optimal results, the distance between cleavage sites should be 40–70 bp apart for the Alt-R Cas9 D10A nickase and 50–70 bp apart for the Alt-R Cas9 H840A nickase. For additional information, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Support section).

  • How far apart should I design my two guides when using the Alt-R® nickases?
    When designing crRNA pairs to use with the Alt-R nickases, we recommend the following:
    • Distance between cleavage sites:
      • Alt-R Cas9 D10A Nickase 3NLS: 40–70 bp
      • Alt-R Cas9 H840A Nickase 3NLS: 50–70 bp
    • PAM-out orientation (right image)
      (light blue = genomic DNA, dark blue = protospacer, and orange = PAM sequence):
    • Pam site orientation (Cas9 nickase experiments)

    For additional information, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Support section).
  • Should I use the Alt-R® Cas9 D10A or H840A nickase?
    It depends upon the experimental design. Cas9 D10A will cut the target strand of DNA (which does not contain the PAM sequence), while Cas9 H840A will cut the non-target strand (which contains the PAM sequence). The availability and location of PAM sites within the target region of DNA needs to be assessed. To learn more about the design and use of Cas9 nickases, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Support section).
  • When should I use the Alt-R® Cas9 nickases, instead of the standard Alt-R S.p. Cas9 Nuclease 3NLS?
    Using the Cas9 nickases can allow improved specificity for generating a double-strand break (DSB) in DNA. Two crRNAs are required to create a DSB using the nickases, one targeting each strand near the target location. This effectively doubles the required recognition sequence from 20 to 40 bases, decreasing the likelihood of an off-target DSB. This makes the Cas9 nickase a good option for experiments requiring a high level of specificity; however, the system can be more challenging to set up, because multiple elements must work together. To learn more about the design and use of Cas9 nickases, refer to the application note at www.idtdna.com/CRISPR-Cas9 (Support section).
  • Do you have GMP versions of the Alt-R® Cas9 nucleases?
    Our catalog options for Alt-R Cas9 nucleases are intended for research use only. For projects requiring GMP materials, now or in the future, contact our experts at CRISPR@idtdna.com to discuss options and see how we can help.
  • How does Alt-R® S.p. HiFi Cas9 Nuclease 3NLS differ from the standard Alt-R S.p. Cas9 Nuclease 3NLS?
    Alt-R S.p. HiFi Cas9 Nuclease 3NLS was developed to reduce off-target cutting events, with similar high, on-target activity as the Alt-R S.p. Cas9 Nuclease 3NLS.

    Alt-R HiFi Cas9 nuclease can be substituted for Alt-R Cas9 nuclease in experiments that are sensitive to off-target cutting events. We recommend testing a dose-response curve using Alt-R HiFi Cas9 or other Cas9 proteins to determine the optimal dosage.
  • When using the Alt-R® Cas9 nickases, do I follow the same protocol as the standard Alt-R S.p. Cas9 Nuclease 3NLS?
    Yes. The delivery of the CRISPR-Cas9 ribonucleoprotein (RNP) complexes with Alt-R Cas9 nickases is similar to RNP with the standard Alt-R S.p. Cas9 Nuclease 3NLS, so the same protocol can be used with these enzymes. However, with the Cas9 nickases, two crRNAs are required to generate a double-strand break, while only one crRNA is required for the standard nuclease.

    When preparing materials to deliver two crRNAs simultaneously, first prepare RNPs with each crRNA separately before RNP delivery. This will help prevent undesireable hybridization between RNAs.
  • Do I need to form my gRNA/RNP complexes separately before multiplexing?
    Yes. Forming the crRNA:tracrRNA:Cas9 ribonucleoprotein complex prior to mixing multiple crRNAs helps prevent undesireable interactions between different crRNAs before they have an opportunity to correctly hybridize with tracrRNA.
  • What is the on-target efficiency of the Alt-R® S.p. HiFi Cas9 Nuclease 3NLS?
    The on-target efficiency of Alt-R S.p. HiFi Cas9 Nuclease 3NLS is typically 90–100% of the standard Alt-R S.p. Cas9 Nuclease 3NLS efficiency at a specific target site. The efficiency of both nucleases varies based upon the target site, cell type, and experimental conditions.
  • What makes Alt-R® S.p. HiFi Cas9 Nuclease 3NLS a high fidelity enzyme?
    Alt-R S.p. HiFi Cas9 Nuclease 3NLS contains proprietary mutations that reduce cutting at problematic off-target sites, while maintaining similar on-target potency.
  • What is the expected reduction in off-target effects (OTEs), when using Alt-R® S.p. HiFi Cas9 Nuclease 3NLS?
    In many cases, Alt-R S.p. HiFi Cas9 Nuclease 3NLS exhibits a 10–20 fold reduction in editing at problematic off-target sites, when compared with the standard Alt-R S.p. Cas9 Nuclease 3NLS. The amount of reduction in off-target editing is highly variable and can differ based on multiple factors, including off-target site, ribonucleoprotein (RNP) dose, and cell type.
  • Do you offer CRISPR crRNA libraries?
    Yes, we offer predesigned CRISPR crRNA libraries and options for creating custom libraries. For more information, please visit www.idtdna.com/customCRISPR.
  • What is the concentration of the Alt-R® Cas9 nucleases and nickases?
    The Alt-R Cas9 nucleases and nickases are stored at concentration of 61 uM (10 mg/mL) in 25 mM Tris-Cl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol (v/v), pH 7.4 .

    These proteins are greater than 95% pure, free of detectable DNase/RNases, absent bacterial DNA (below the limit of detection), and contain less than 10 EU/mL endotoxin.
  • When using the Alt-R® S.p. HiFi Cas9 Nuclease 3NLS, do I follow the same protocols for the standard Alt-R S.p. Cas9 Nuclease 3NLS?
    Yes. The Alt-R S.p. HiFi Cas9 Nuclease 3NLS can be directly substituted for the standard Alt-R S.p. Cas9 Nuclease 3NLS in the protocols.
  • How many experiments can I expect to get from a tube of Cas9?

    It depends upon your method for delivery, reaction volume, and Cas9 concentration.

    As an example:

    Tube size Delivery method Reaction volume Cas9 concentration Number of reactions
    100 µg Lipofection 150 µL 10 nM ~400
    100 µg Electroporation 10 µL 3 µM ~20
  • How do you recommend assessing off-target effects?
    To avoid problems with coverage using in-cell techniques, we recommend initial screening for off-target effects using cell-free methods, such as circle-seq or site-seq. Guide-seq is one unbiased live-cell method to consider. Bioinformatics-based methods for predicting off-target sites are not sufficiently comprehensive.
  • I cannot see CRISPR-Cas9 editing. What can I do?
    • Double-check that the your crRNA sequence matches the species and target location as expected. For example, verify that the target site in your cells does not show any polymorphism that could affect the potency of the crRNA.
    • Confirm that you can observe successful editing using a known positive control guide RNA, such as the Alt-R® positive control crRNAs for human, mouse, or rat.
    • If the positive controls work but the experimental crRNA does not, test additional target sites nearby to see if a more efficient guide RNA can be found.

    For additional support, contact our experts at applicationsupport@idtdna.com.
  • What reagents will I need in addition to the Alt-R® RNAs for a CRISPR-Cas9 genome editing experiment?

    Ribonucleoprotein (RNP) components

    The Alt-R® CRISPR-Cas9 System includes a target-specific CRISPR-Cas9 crRNA and a universal CRISPR-Cas9 tracrRNA, both of which are required for genome editing using the Alt-R CRISPR-Cas9 System protocol. Nuclease-Free Duplex Buffer, which is used to complex the crRNA and the tracrRNA, is included with the Alt-R tracrRNA. You will also need an S. pyogenes Cas9 endonuclease:

    • Alt-R S.p. Cas9 Nuclease 3NLS, recommended for most experiments
    • Alt-R S.p. HiFi Cas9 Nuclease 3NLS, for reduced off-target effects
    • Alt-R S.p. Cas9 D10A or H840A Nickase 3NLS, for increased specificity and reduced off-target effects
    • S.p. Cas9 Expression Plasmid.

    Delivery

    Supply your own transfection reagents to effectively deliver CRISPR reagents into your cells. We recommend delivery of an RNP for best results. The CRISPR-Cas9 System is compatible with transfection (lipid-mediated or electroporation) and microinjection.

    Additional reagents

    We also highly recommend the use of control crRNAs, which can be ordered as part of the Alt-R CRISPR-Cas9 Control Kits or individually.

    The Alt-R Cas9 Electroporation Enhancer, which is a non-targeting carrier DNA, can increase the efficiency of electroporation. The fluorescently labeled Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550, which functions like the unlabeled Alt-R CRISPR-Cas9 tracrRNA, can be used to monitor transfection efficiency, study cellular localization, or enrich for CRISPR-edited cells.

    If you are interested in additional options for genome editing, we also offer reagents to perform CRISPR-Cpf1 genome editing.

  • How should I store the Cas9 protein?

    All Alt-R® S.p. Cas9 nucleases and nickases should be stored at –20°C. Under optimal storage conditions, the protein maintains functionality for at least 2 years.