TechVault
  • I transfected my cells with the fluorescently labeled Alt-R® CRISPR-Cas9 tracrRNA – ATTO™ 550. When should I sort my cells by fluorescence activated cell sorting (FACS)?

    For optimal results, we recommend sorting your cells by FACS 24 hours after delivery of the ribonucleoprotein (RNP) complex containing the Alt-R® CRISPR-Cas9 tracrRNA – ATTO™ 550. The optimal signal intensity occurs at 24 hours, after which signal intensity begins to decrease.

  • What is Alt-R® Cas9 Electroporation Enhancer?

    Alt-R® Cas9 Electroporation Enhancer functions as a carrier DNA and is a single-stranded DNA oligonucleotide that was computationally designed to be non-homologous to human, mouse, or rat genomes.


  • Can I use a CRISPR-Cas9 crRNA protospacer sequence that is shorter than 20 nt?

    The Alt-R® CRISPR-Cas9 crRNA ordering tool (accessible at www.idtdna.com/CRISPR-Cas9) accommodates 19 and 20 nucleotide (nt) protospacer sequences; however, we recommend 20 nt sequences for most experiments. Other formats can be ordered as custom RNAs.

    There are reports in the literature suggesting that CRISPR-Cas9 nuclease specificity can be improved through use of truncated guide RNAs [1]. For example, 17 nt protospacer elements have been reported to reduce off-target effects.

    In contrast, our research investigating the effect of shorter protospacer element length on CRISPR-Cas9 nuclease specificity demonstrated that 20 nt protospacer elements were optimal, with 19 nt protospacers providing similar strong editing efficacy in most cases (see figure). When using Alt-R S.p. HiFi Cas9 Nuclease, 20 nt protospacer sequences provide the greatest amount of genomic editing (data not shown).

    Figure. 20 nt protospacer element provides optimal genome editing. crRNAs with varying protospacer element lengths (17–20 nt) were designed to 12 distinct HPRT target sites. crRNA:tracrRNA complexes were reverse transfected into a HEK-293 cell line stably expressing S. pyogenes Cas9, using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher). Genomic DNA was isolated and editing was measured by PCR amplification of target sites followed by cleavage with T7EI mismatch endonuclease (Alt-R® Genome Editing Detection Kit) and analysis using the Fragment Analyzer™ (Advanced Analytical). At all but one of the 12 target sites, crRNAs with 19 and 20 base protospacer elements provided the greatest amount of genomic editing.

    Reference

    1. Fu Y, Sander JD, et al. (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotech, 32(3):279–284.
  • Can Alt-R® Cas9 Electroporation Enhancer negatively impact the activity of my guide RNA?

    We have not found the enhancer to negatively impact guide RNA performance. There was no reduction in transfection efficiency in the presence of the enhancer. However, the amount of improvement in electroporation efficiency can vary.


  • How much Alt-R® Cas9 Electroporation Enhancer should I use during delivery of the ribonucleoprotein (RNP) complex?

    We see dose response curves that vary for different guide sequences, depending on the potency of the guide sequence. We recommend titrating the amount of RNP for each cell type of interest.

    Start with a 1:1 ratio of enhancer:RNP and optimize using a titration gradient of the RNP. For the Amaxa® Nucleofector® System (Lonza), we recommend 4 µM of Alt-R® Cas9 Electroporation Enhancer, and for the Neon® System (Thermo Fisher Scientific), we recommend 1.8 µM of Alt-R Cas9 Electroporation Enhancer.


  • How much fluorescently labeled tracrRNA should I use in my CRISPR-Cas9 experiments?

    The amount of Alt-R® CRISPR-Cas9 tracrRNA – ATTO™ 550 needed varies by application and transfection method. Here are our recommendations. Use these as starting points as you optimize your experiments.

    Table. Final concentration of Alt-R® CRISPR-Cas9 tracrRNA – ATTO™ 550 to use in transfection

     FACS analysisFluorescence microscopy
    Lipofection~10 nM10 nM
    Electroporation≥0.5 µM
    ~0.5 µM
  • What CRISPR-Cpf1 crRNA controls should I use?

    We have recommended positive and negative control sequences for Cpf1 crRNAs available for ordering on the website. These recommended sequences can be found in the Alt-R® CRISPR Cpf1 user guides (accessible at www.idtdna.com/CRISPR-Cpf1, Support section), and are also available on the side bar of the CRISPR-Cpf1 crRNA ordering pages (accessible at www.idtdna.com/CRISPR-Cpf1).


  • I am seeing cell death after transfection in my CRISPR-Cas9 experiments. What can I do to prevent this?

    If you are using transfection reagents:

    Transfection reagents can be cytotoxic, and the extent of cell death can vary from one cell type to the next. If you are seeing excessive cell death, the first step should be to optimize the transfection reaction using our Alt-R® CRISPR-Cas9 HPRT Positive Control crRNA, which is available for human, mouse, and rat cells. The goal is to use the least amount of lipid transfection reagent, without significantly affecting transfection efficiency.

    You could try a different transfection reagent, as minor differences in chemistry can impact toxicity. Make sure to look for transfection reagents optimized for RNA to transfect the Alt-R CRISPR-Cas9 RNAs and RNPs. We have had success using TransIT® Transfection Reagent (Mirus Bio) for transfection of the S.p. Cas9 Expression Plasmid and RNAiMAX™ Reagent (Thermo Fisher Scientific) for the Alt-R CRISPR-Cas9 RNAs in HEK-293 cells. Transfection reagents for use in other cell lines should be empirically optimized.

    If you are doing electroporation:

    Optimal electroporation conditions vary by cell line as well as by type, amount, and size of material (e.g., plasmid, RNA, DNA, or ribonucleoprotein) being introduced into the cells. Examples of electroporation conditions that may need optimization include number of cells, relative amounts of materials, voltage, pulse width, and number of pulses. For additional information, see Successful CRISPR genome editing in hard-to-transfect cells.

    Could your targeted gene be essential for cell viability?

    It is also possible that your target gene is vital to your cells and, thus, its knockout results in cell death. If the positive control (Alt-R CRISPR-Cas9 Human HPRT Positive Control crRNA) works and your target is knocked out, it is likely you are working with a vital gene. In this case, consider RNAi methods to knock down expression of your target gene [visit our Dicer-substrate short interfering RNA (DsiRNA) webpage for additional information].


  • There are no NGG PAM sequences close to my locus of interest. What can I do?

    If the region of interest is so narrow that there are no “GG” motifs, you may have to explore Cas endonucleases from other organisms that recognize different PAM sites.

    Jiang et al. have shown that S. pyogenes Cas9 also recognizes another PAM site: NAG, though with lesser efficiency than NGG [1].

    Alternatively, for T-rich sequences, the Cpf1 system may be of utility, as it recognizes a TTTV PAM site, where V is A, C, or G. IDT offers the Alt-R® Cpf1 System (crRNA, nuclease, and electroporation enhancer), which is beneficial for editing around T-rich sequences. Visit the Alt-R Cpf1 System webpage for more information, or contact us at applicationsupport@idtdna.com to discuss what we have learned in our ongoing research.

    Reference

    1. Jiang W, Bikard D, et al. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotech, 31:233–239.
  • What is Alt-R® Cpf1 Electroporation Enhancer?

    Alt-R® Cpf1 Electroporation Enhancer functions as a carrier DNA and is a single-stranded DNA oligonucleotide that was computationally designed to be non-homologous to human, mouse, or rat genomes. The use of this enhancer is important for efficient electroporation of ribonucleoproteins (RNPs), which in turn is important for increased rates of editing.


  • How much Alt-R® Cpf1 Electroporation Enhancer should I use?

    We have observed that dose response curves vary for different guide sequences, depending on potency of the guide. We recommend titrating the amount of RNP and keeping the amount of enhancer fixed.

    For the Amaxa® Nucleofector® System (Lonza), we recommend 4 µM of Alt-R® Cpf1 Electroporation Enhancer, and for the Neon® System (Thermo Fisher Scientific), we recommend 1.8 µM of Alt-R Cpf1 Electroporation Enhancer. Toxicity may be observed at high concentrations of enhancer.


  • I cannot see CRISPR-Cpf1 editing. Why?

    Ensure that you have included the Alt-R™ Cpf1 Electroporation Enhancer (carrier DNA) in the electroporation and that you are using one of our positive control crRNAs to demonstrate efficient delivery.

    Verify that the target site in your cells does not show any polymorphism that could affect the potency of the crRNA.

    Target 3 or more PAM sites in your gene of interest to identify a site that provides optimal editing efficiency, because we have found that approximately 50% of the Cpf1 PAM sites give detectable editing.


  • If the target area does not contain a protospacer adjacent motif (PAM) sequence, will the CRISPR-Cas9 complex bind?

    No, CRISPR-Cas9 complexes do not recognize, or recognize extremely poorly, targets lacking PAM sequences [1].

    The PAM sequence recognized by the S. pyogenes CRISPR-Cas9 system is NGG. If this sequence is not present in your target, you may be able to use other CRISPR systems (from other bacterial species) that recognize different PAM sequences. The following table lists examples of PAM sequences:

    Bacterial species PAM sequence Reference
    S. pyogenes (Cas9)* NGG [1]
    Acidaminococcus sp BV3L6 (Cpf1)* TTTV [2]
    Streptococcus thermophilus NNAGAA
    NGGNG
    [3,4]
    Neisseria meningitidis NNNNGATT [3,4]

    * In addition to the Alt-R® CRISPR-Cas9 System, we offer the Alt-R CRISPR-Cpf1 System (crRNA, nuclease, and electroporation enhancer). Note that each CRISPR nuclease has unique requirements for crRNA and electroporation enhancer design. You will not be able to use Cas9 and Cpf1 reagents in the same reaction.

    References

    1. Hsu PD, Scott DA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31(9):827–832.
    2. Zetsche B, Gootenberg JS, et al. (2015) Cpf1 is a Single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell, 163(3):759–771.
    3. Esvelt KM, Mali P, et al. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods, 10(11):1116–1121.
    4. Ran FA, Hsu PD, et al. (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8(11):2281–2308.

  • My CRISPR-Cas9 genome editing efficiency seems very low. How can I improve my experiments?

    Possible reasons for low editing efficiency

    • Depending on your method of assessment, your editing efficiency may be underrepresented. Mismatch endonucleases like T7EI do not detect single base changes, and under-estimate actual editing compared to direct sequencing.
    • Not every sequence associated with a PAM site performs the same. For example, polymorphisms in the protospacer binding site may reduce editing efficiency. Base mismatches also become more detrimental to editing the closer they are to the PAM site.

    Recommendations

    • Try 2 or 3 different PAM sites in your gene of interest to identify a site that provides optimal editing efficiency.
    • Include control experiments to help monitor or optimize CRISPR reagent delivery. Alt-R® CRISPR-Cas9 HPRT Positive Control cRNAs and Alt-R CRISPR-Cas9 Negative Control cRNAs are available for human, mouse, and rat.
    • If you are using electroporation methods, including Alt-R Cas9 Electroporation Enhancer could improve transfection efficiency and, therefore, editing efficiency.
    • Please contact us at applicationsupport@idtdna.com for additional assistance; having the results of your control experiments available will facilitate our ability to help you.
  • Is it possible for the Alt-R® Cpf1 Electroporation Enhancer to integrate at the Cpf1 cut site?

    Our next generation sequencing data demonstrated a very low rate of integration of the enhancer into the target site (fewer than 1 in 10,000 reads, which is much lower than the insertion rate of random pieces of cellular DNA). Therefore, enhancer integration should not negatively impact experimental outcomes.


  • Can the Alt-R® Cpf1 Electroporation Enhancer negatively impact the activity of my guide?

    We have not found the electroporation enhancer to negatively impact guide performance. There was no reduction in transfection efficiency in the presence of the enhancer. However, the amount of improvement in transfection efficiency can vary but, in many cases, is important for successful genome editing.


  • How should I deliver the CRISPR-Cpf1 components into cells?

    We recommend delivering a ribonucleoprotein (RNP), consisting of Cpf1 nuclease pre-complexed to crRNA. Non-targeting carrier DNA (i.e., Alt-R® Cpf1 Electroporation Enhancer) should also be included in the electroporation for efficient editing.

  • How should I store the Cpf1 protein?

    Alt-R® A.s. Cpf1 Nuclease 2NLS should be stored at –20°C. Under optimal storage conditions, the protein maintains functionality for at least 2 years.


  • Is the Alt-R® CRISPR-Cpf1 ribonucleoprotein (RNP) complex stable? Do I need to make a fresh complex for each experiment?

    Stability studies for the Alt-R® Cpf1 RNP complex are currently ongoing, so we recommend making fresh RNP complexes for each experiment. For additional information, please contact applicationsupport@idtdna.com.

  • What is the protospacer adjacent motif (PAM) sequence for Cpf1? How is this different from Cas9?

    The PAM sequence for the Cpf1 system is TTTV [1], where "V" is a A, C, or G. The Cpf1 PAM sequence can be advantageous when working with T-rich target sequences. In contrast, the Cas9 PAM sequence is NGG. 

    Reference

    1. Zetsche B, Gootenberg JS, et al. (2015) Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell, 163(3):759–771.