Genome Editing
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The key to successful electroporation in CRISPR genome editing experiments

Use a carrier DNA, like the Alt-R™ electroporation enhancers

Increasing evidence shows that the most efficient CRISPR-based genome editing results from delivery of CRISPR reagents as ribonucleoprotein (RNP) complexes, instead of expression plasmids or in vitro transcribed RNA (see data) [1]. Many scientists are able to use lipofection methods to transfect RNPs into their cultured cells. However, electroporation is required for some cell types that are refractory to lipid-mediated transfection, or that are susceptible to cytotoxicity from the lipid reagents.

As part of our extensive research into using electroporation in CRISPR experiments, IDT scientists have developed 2 electroporation enhancers that are specific for either the Alt-R® CRISPR-Cas9 System or the Alt-R CRISPR-Cpf1 System. Both enhancers are composed of single-stranded, carrier DNA, optimized to work with the Amaxa® Nucleofector® device (Lonza) and Neon® System (Thermo Fisher) to increase electroporation efficiency and thereby increase genome editing efficiency. The enhancer sequences were specifically designed to avoid homology with human, mouse, and rat genomes.

Note that Alt-R Cas9 and Cpf1 Electroporation Enhancers are sequence-optimized for each nuclease, and therefore, are not interchangeable between systems. A quick comparison of CRISPR genome editing using Cas9 or Cpf1 is available at www.idtdna.com/CRISPR.

  • Alt-R® Cas9 Electroporation Enhancer—improve editing efficiency
  • The CRISPR-Cas9 system is the gold standard for CRISPR-based genome editing. If your cells require electroporation, use the Alt-R Cas9 Electroporation Enhancer to improve editing efficiency (Figure 1). The optimal amount of enhancer to include will differ with electroporation instrument.

    The amount of improvement in editing efficiency also will vary by cell type. In some cases, use of the Alt-R Cas9 Electroporation Enhancer allows you to decrease the amount of Cas9 RNP required for optimal editing efficiency (Figure 1). A reduction in RNP is advantageous because of possible improvements in cell survival and lowered risks of potential off-target editing.

    IDT protocols and customer methods are available on the Support tab of www.idtdna.com/CRISPR-Cas9.

    Alt-R Cas9 Electroporation Enhancer improves editing efficiency

    Figure 1. Alt-R® Cas9 Electroporation Enhancer improves CRISPR editing efficiency in ribonucleoprotein (RNP) electroporation experiments. K562 (A), Jurkat (B), and HEK-293 (C) cells were transfected (Amaxa® System, Lonza) with 0.125–4 µM RNP (Alt-R S.p. Nuclease 3NLS complexed with Alt-R CRISPR-Cas9 crRNA and tracrRNA). Electroporation reactions were performed in the presence (dark blue) or absence (light blue) of 4 µM Alt-R Cas9 Electroporation Enhancer. Editing efficiency was determined 48 hr after electroporation using the Alt-R Genome Editing Detection Kit (IDT), which provides the major components required for T7EI assays.


  • Alt-R® Cpf1 Electroporation Enhancer—required for efficient genome editing
  • The Alt-R CRISPR-Cpf1 System allows you to target AT-rich regions. Because the Cpf1 RNP does not transfect easily, the electroporation enhancer is required for genome editing experiments (Figure 2). The optimal amount of enhancer to include will differ by electroporation instrument.

    IDT protocols are available at www.idtdna.com/CRISPR-Cpf1.

    Alt-R Cpf1 Electroporation Enhancer is required for optimal editing efficiency

    Figure 2. Alt-R™ Cpf1 Electroporation Enhancer is required for efficient CRISPR editing in ribonucleoprotein (RNP) electroporation experiments. HEK-293 cells were transfected with 5 µM RNP (Alt-R A.s. Cpf1 Nuclease 2 NLS complexed with Alt-R CRISPR-Cpf1 crRNA) as instructed in the Alt-R CRISPR-Cpf1 User Guide—RNP electroporation, Amaxa® Nucleofector® system. 12 Cpf1 PAM sites in the HPRT gene were targeted by Alt-R CRISPR-Cpf1 crRNAs. The electroporation reactions contained either no (dark blue) or 3 µM (light blue) Alt-R Cpf1 Electroporation Enhancer. Editing efficiency was determined 48 hr after electroporation using the Alt-R Genome Editing Detection Kit, which provides the major components required for T7EI assays. PAM = protospacer adjacent motif (Cpf1 PAM sequence is TTTV); x-axis: numbers specify gene locations; S = sense strand; AS = antisense strand.


Reference

  1. Kim S, Kim D, Cho SW. (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res, 24:1012–1019.

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

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.

Successful CRISPR genome editing in hard-to-transfect cells (i.e., Jurkat cells)—Use the conditions presented here for Clone E6-1 Jurkat cells as a starting point for optimization of CRISPR reagent delivery in cell types requiring electroporation.

CRISPR genome editing: 5 considerations for target site selection—Learn how your genome editing experiments can be improved with 5 quick tips for target selection and with reagents from 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.

© 2017 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