Verification of gene editing efficiency

  • Before proceeding with downstream applications, verify the gene editing efficiency of the control target and select the condition that shows the highest level of editing efficiency in future screening experiments.
  • To estimate the CRISPR-Cas9-mediated editing efficiency in a pooled cell population, use the GeneArt Genomic Cleavage Detection Kit, or perform Ion Torrent next generation sequencing or a Sanger sequencing–based analysis.
  • While the genomic cleavage detection (GCD) assay provides a rapid method for evaluating the efficiency of indel formation following an editing experiment, next-generation sequencing (NGS) of the amplicons from the edited population or Sanger sequencing of amplicons cloned into plasmids give a more accurate estimate of the percent editing efficiency and indel types.
     

GeneArt Genomic Cleavage Detection (GCD) Assay

  • After transfections, use the GeneArt Genomic Cleavage Detection Kit to estimate the CRISPR-Cas9-mediated cleavage efficiency in a pooled cell population.
  • You can design and order target-specific primer sets for the GCD assay through our GeneArt CRISPR Search and Design tool, available at thermofisher.com/crisprdesign.
  • To perform the GCD assay for the positive control, you need the primers listed in Table 1. We recommend using Invitrogen Custom DNA Value or Standard Oligos, available from thermofisher.com/oligos, for target specific primer sets needed for the GCD assay.
  • You can set up the GCD assay in a 96-well plate format and analyze multiple gRNA-treated samples in parallel on a 2% E-Gel 48 agarose gel (48‑well).
  • Pick the clones that show the highest cleavage efficiency to use in your experiments. Note that the clone that shows the highest cleavage efficiency may not always be the clone with the highest expression.
  • For more information and detailed protocols, see the GeneArt Genomic Cleavage Detection Kit User Guide.

 

Table 1. Target sequences for the positive and negative control (non-targeting) TrueGuide Synthetic gRNA sequences.

TrueGuide Synthetic Guide RNA Controls* Primers for the GeneArt Cleavage Detection (GCD) Assay
Locus Target-specific crRNA sequence Forward GCD primer Reverse GCD primer
Human AAVS1** 5’-GCCAGUAGCCAGCCCCGUCC-3’ 5’-GAATATGTCCCAGATAGCAC-3’ 5’-GTTCTCAGTGGCCACCCTGC-3’
Human HPRT (ln)** 5’-GCAUUUCUCAGUCCUAAACA-3’ 5’-ACATCAGCAGCTGTTCTG-3’ 5’-GGCTGAAAGGAGAGAACT-3’
Human CDK4† 5’-CACUCUUGAGGGCCACAAAG-3’ 5’-GCACAGACGTCCATCAGCC-3’ 5’-GCCGGCCCCAAGGAAGACTGGGAG-3’
Mouse Rosa 26** 5’-CUCCAGUCUUUCUAGAAGAG-3’ 5’-AAGGAGCGAGGGCTCAGTTGG-3’ 5’-GGTGAGCATGTCTTTAATCTACCTCG-3’
Negative control (non-targeting) 5’-AAAUGUGAGAUCAGAGUAAU-3’ N/A N/A
*Available in TrueGuide Synthetic sgRNA and crRNA formats (see thermofisher.com/trueguide). **Specific to an intron. †Targets 5' exons.

 

Sequence analysis

  • For Sanger sequencing-based editing efficiency analysis, refer to our application note referenced at thermofisher.com/sangercrispr.
  • If you are experienced in next-generation sequencing (NGS) and analysis, you can use barcoded target-specific amplicon primers and perform multiplex analysis using several gRNA-treated samples in parallel. Multiplex analysis using NGS is especially useful when using the custom arrayed plate format for TrueGuide Synthetic gRNA transfections. For more information on NGS analysis, refer to Ion Torrent targeted sequencing solutions at thermofisher.com/ionapliseqsolutions.