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CRISPR/Cas9 Sequencing

 
 
Use CRISPR/Cas9 sequencing to:
  • Verify your guides library by deep sequencing
  • Validate CRISPR/Cas9 targets and mutation efficiency by amplicon deep sequencing
  • Discover the candidates with most impact from screenings and the frequency and implications of the edits

 

Overview

Considerations before starting a CRISPR/Cas9 sequencing project:

  • Minimal required sequencing length?
  • Sequencing depth?
  • Analysis – frequency versus mutation implication?
  • Functional setup (replicates and conditions)?

Let us guide you – from design to analysis

Example projects using CRISPR/Cas9 sequencing:

  • Verification of CRISPR/Cas9 plasmid libraries
  • Analysis of target loci
  • Analysis of predicted off-target loci

Applications related to CRISPR/Cas9:

  • Amplicon deep sequencing
  • RNA sequencing
  • Eukaryotic resequencing

Workflow

 
A typical workflow for a CRISPR/Cas9 sequencing project is shown in the graphic below. Please note that our highly-modular processes allow you various entry and opting out options. If you outsource your entire NGS project to Microsynth or only parts of it is up to you.
 

For further reading and a detailed technical description, please download our Application Note Illumina CRSIPR/Cas9 sequencing (see related downloads).

Results

Our gene-editing analysis module addresses the analysis of experiments where a certain gene is edited by molecular biological techniques such as CRISPR/Cas9. The module is designed to help with both, pre- and post-editing analysis. You can analyze your plasmid library containing the sgRNAs as well as the target genes after the gene editing took place. Answers to the following major questions will be provided:

  1. Which sgRNAs are present in the plasmid library and what is their frequency? (see Figure 1)
  2. Was the gene editing event successful and if so what was the effect on the target gene in comparison to its reference sequence? (see Figure 2+3 and Table 1)

The analysis of the plasmid library requires no references and as results the sequenced sgRNAs are provided along with their frequencies.

For post editing analysis the edited genes are amplified, sequenced and aligned against the reference sequence. Thus insertions and deletions (InDels) are identified and reported along with their frequencies.

 

Figure 1: Detail of FASTA file containing unique sequences annotated with expected sequencing error and sequence frequency.

Figure 2: Histogram of deleted positions in comparison to the unaltered reference sequence.

Figure 3: Histogram of deletion sizes.

 
Table 1: Detail of a results table listing InDel structure of the dereplicated sequences along with their frequencies within the dataset.