CRISPR Sequencing

CD Genomics provides CRISPR-Cas9 knockout validation and potential off-target detection in a high-throughput and cost-effective manner by harnessing advanced next generation sequencing (NGS). Our team members have experience in both genome editing and NGS, enabling extensive support to your research.

What is CRISPR-Cas9 system?

The CRISPR-Cas9 gene targeting system, requiring two components: a custom guide RNA (sgRNA, consisting of target-specific crRNA sequence and tracrRNA) and a non-specific CRISPR-associated endonuclease (Cas9), is a new genome engineering tool that enables researchers to edit parts of the genome by removing, adding or altering a section of the DNA sequence in organism. It is currently the simplest, most versatile, precise and effective method of genetic manipulation that has many potential applications including medicine and crop seed enhancement. CRISPR-Cas9 has also been adapted to enable high-throughput genome editing and has revolutionized the generation of targeted mutations.

Introduction to CRISPR sequencing

Validating edits is especially important in the CRISPR experimental process. Next-generation sequencing (NGS) is an easy and high-throughput method to check for desired mutations since off-target mutation and not an edit in the target gene could happen. CRISPR amplicon sequencing has become a standardized validation technique for academia, clinic, and industry. High-throughput CRISPR screening is based on the principle of targeted amplicon sequencing by conducting PCR experiments using primers flanking the target region. Targeted amplicon sequencing is the most sensitive method for checking mutations and the detection frequencies are as low as 0.01%.

To address the emerging needs of research communities, CD Genomics has developed an affordable, reliable, and high-throughput strategy for screening and validating CRISPR-Cas9 based mutations by harnessing amplicon based next-generation Sequencing. Our CRISPR next-generation sequencing service can give you direct and detailed information about the nature and diversity of the mutations, including confirmation of knockout/knockin alleles, assessment of sgRNAs cutting efficiency, homozygous and heterozygous identification, mutation frequencies calculation et al.

CRISPR Sequencing  Workflow

Our highly experienced expert team executes quality management following every procedure to ensure comprehensive and accurate results. Our CRISPR mutation sequencing workflow is outlined below, including DNA isolation, PCR experiments, deep sequencing, and data analysis. Our CRISPR mutation sequencing enables researches to validate guides library, validate CRISPR/Cas9 targets and mutation efficiency, as well as discover the most promising target sites.

Figure 1. Our workflow of CRISPR amplicon sequencing.

Service Specifications

	Sample requirements and preparation Samples types: cell or DNA samples after CRISPR-Cas gene editing manipulation DNA sample: ~1 μg (concentration ≥ 30 ng/μl; OD260/280=1.8~2.0) Cell sample: 1×106 cells or 10 cm2 cell culture The sample preparation procedures cover DNA isolation, purification, quantification, QC, etc.
	Deep sequencing Illumina HiSeq platforms, paired-end 150 bp or 300 bp; PacBio’s SMRT platform Sequencing depth: ≥ 1000x Analysis of sequencing quality metrics
	Bioinformatics Analysis Raw data QC Reference alignment Validation of CRISPR libraries Analysis of target loci Analysis of predicted off-target loci

• Extensive multiplexing flexibility and high-throughput sequencing, up to 10⁴ samples per run
• Ultra-deep sequencing of amplicons or captured regions, in excess of 1000X coverage
• Cost-effective, and highly sensitive detection levels without bias
• No need for laborious and time-consuming cloning steps
• Dedicated support from specialized Ph.D.-level scientists

CD Genomics is able to help you to detect the potential off-target effects via targets enrichment and deep sequencing at fairly inexpensive prices. We can sequence hundreds of samples simultaneously. The InDel distribution pattern will be analyzed and mutation can be confirmed with the CRISPResso tool. If you have additional requirements or questions, do not hesitate to contact us.