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 the Performance section of www.idtdna.com/CRISPR-Cas9) . 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 here.
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 Cas9 protocols and customer methods are available under Resources at www.idtdna.com/CRISPR-Cas9.
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 Cpf1 protocols are available under Resources at www.idtdna.com/CRISPR-Cpf1.
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.