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CRISPR genome editing: 5 considerations for target site selection

Use the Alt-R® CRISPR-Cas9 System to increase target editing efficiency

CRISPR-Cas9 genome editing has revolutionized genomics research and will continue to do so for years to come. With a high-quality S. pyogenes Cas9 nuclease and optimized crRNA and tracrRNA, the Alt-R® CRISPR-Cas9 System from IDT has all the reagents needed for successful genome editing, and offers numerous advantages over alternative methods. These include:

  • Higher on-target editing potency than other CRISPR-Cas9 systems

  • Efficient delivery of the Cas9 ribonucleoprotein (RNP) with lipofection or electroporation

  • No toxicity or innate immune response activation,as observed with in vitro transcribed mRNA and sgRNAs

Effective genome editing, even when done using an optimized system, can be difficult. To ensure the best results, consider these points on target selection as you design your experiments.

  1. Does your target gene express multiple transcripts?

  2. Due to alternative exon splicing, a single gene can express multiple transcripts, or splice variants. If your target gene expresses splice variants which do not contain your target exon, your gene may retain activity in knockout experiments. Use Ensembl (or other genome databases) to determine whether multiple transcripts exist for your target gene. If so, design the crRNA such that the exon you are targeting is present in every transcript. See Figure 1 for examples of alternatively spliced transcripts to consider.

    Constitutive and alternate exon splicing

    Figure 1. Constitutive and alternative exon splicing. In constitutive splicing, each exon is joined together in the order it occurs in nuclear RNA (pre-mRNA). Alternative splicing of these exons can produce a variety of mRNA isoforms. The 5 possible alternative splicing events that produce them are: exon skipping, mutually exclusive exons, alternative splice-site selection (5′ and 3′), and intron retention. For an individual pre-mRNA, individual exons can exhibit different types of alternative splicing patterns [1].


  3. Are there SNPs present in your target site?

  4. When single nucleotide polymorphisms (SNPs) are present in your target sequence, the protospacer element of your crRNA may have difficulties binding to it (Figure 2). This can ultimately lead to inefficiencies in genome editing. Check reliable databases, such as the NCBI dbSNP or UCSC Genome Browser, to ensure your target sequence does not contain SNPs.


    Figure 2. Components of the Alt-R® CRISPR-Cas9 System for directing Cas9 endonuclease to genomic targets. The cleavage site is specified by the protospacer element of the crRNA (thick green bar). The crRNA protospacer element recognizes 19 or 20 nt on the opposite strand of the NGG PAM site.

    Alt-R® CRISPR-Cas9 System performance

    Research by IDT scientists has shown that the Alt-R CRISPR-Cas9 System provides the highest percentage of on-target genome editing when compared to competing designs, including both native S. pyogenes crRNA:tracrRNA and single fusion sgRNA triggers. For details on this research, watch our webinar: Increase efficiency of genome editing with the Alt-R® CRISPR-Cas9 System: Design and use.

  5. What is the ploidy of your cells?

  6. The existence of multiple alleles will inevitably lead to an increase in the number of possible editing events. For this reason, it is important to take your cells’ ploidy into account, especially when working with triploid or tetraploid cell lines or organisms. While T7EI mismatch endonuclease assays can be effective in estimating editing efficiency [2], DNA sequencing is the recommended method for identifying the type of editing event(s) that took place, whether in diploid or multiploid cells.

  7. Is there a known phenotype associated with the targeted gene?

  8. If you are unaware of your target gene’s associated phenotype, you may wrongly attribute your results to incorrect causal factors. For example, successful genome editing experiments that result in lethality, proliferation, or differentiation can be easily misinterpreted as errors in methodology. Being familiar with expected phenotypic effects will eliminate this confusion and lead to more meaningful results.

  9. Are you selecting for monoclonal populations?

  10. If you plan to select clonal populations that contain specific editing events, we recommend checking the editing efficiency of the entire cell population first. By doing this, you can assess the chances of picking individual clones. Also, if you are making limited dilutions, do so as soon as possible after your editing experiment, as non-edited cells could potentially outgrow edited cells.

For more information about the Alt-R CRISPR-Cas9 System from IDT, watch our latest webinar: Alt-R® CRISPR-Cas9 System: Ribonucleoprotein delivery optimization for improved genome editing.

Still looking more information? Visit the Alt-R CRISPR-Cas9 System webpage, or contact us at applicationsupport@idtdna.com.

References

  1. Cartegni L, Chew SL, Krainer AR. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet, 3(4):285–298.

  2. Vouillot L, Thélie A, Pollet N. (2015) Comparison of T7EI and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3: Genes|Genomes|Genetics, 5(3):407–415.

Product focus—genome editing with Alt-R® CRISPR Reagents

Alt-R CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precision control with delivery of Cas9 ribonucleoprotein (RNP)
  • Efficient delivery of the RNP with lipofection or electroporation
  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System.


Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System allows for new CRISPR target sites that are not available with the CRISPR-Cas9 System, and produces a staggered cut with a 5′ overhang. These reagents:

  • Enable genome editing in organisms with AT-rich genomes
  • Allow interrogation of additional genomic regions compared to Cas9
  • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
  • Permit efficient delivery of the RNP into cells by electroporation

Learn more about the Alt-R CRISPR-Cpf1 System.


CRISPR support tools

Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.

Find out more about IDT’s entire line of CRISPR products.

Additional resources

CRISPR guide RNA format affects genome editing outcomes—Learn how use of different formats for the guide RNAs associated with CRISPR-Cas9 genome editing can lead to different editing outcomes. The optimized, short RNA oligos that make up the crRNA and tracrRNA components of the Alt-R® CRISPR-Cas9 System outperform other CRISPR guide RNA formats. Unlike DNA expression constructs, short RNA oligos are unable to incorporate into the target genome, resulting in cleaner editing.

6 pieces of data that will change how you set up your CRISPR-Cas9 experiments—Discover how you can improve the efficiency of CRISPR-Cas9 genome editing. IDT scientists evaluated several factors that influence how we design and perform genome editing experiments. Review the data and results for 6 important factors that were addressed. These experimental findings resulted in a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.

Getting started with Alt-R® CRISPR-Cas9 genome editing—Webinar: Watch a recording of our webinar to learn about the components of the Alt-R™ CRISPR-Cas9 System, get information on designing Alt-R CRISPR crRNA oligos, and review the genome editing protocol from the user guide.

Authors: Rolf Turk, PhD, is a staff scientist, and Nolan Speicher is a scientific writer, both at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


CRISPR-Cas9 Genome Editing

With Alt-R CRISPR-Cas9 System get the core components for improved on-target editing performance, while experiencing lower toxicity, and no innate cellular response.

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Related Content

Product page:

Alt-R CRISPR-Cas9 System

Webinar:

New RNA tools for optimized CRISPR/Cas9 genome editing

Learn how research conducted at IDT led to the development of a potent new set of CRISPR-Cas9 genome editing tools.

Poster:

Quantitative Measurement of CRISPR/Cas9 Gene Editing at the Level of Genomic DNA for sgRNA Site Selection Algorithm Development

User guide:

Alt-R CRISPR-Cas9 System User Guide