Accelerate CRISPR genome editing using next-generation sequencing

NGS enables researchers to have complete control throughout CRISPR-Cas9 genome editing experiments

CRISPR Genome Editing and NGS

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing is a revolutionary method in which a programmable RNA targets a nuclease (eg, Cas9) to a specific location in the genome.1,2 The speed, simplicity, and precision with which CRISPR-Cas9 technology enables genetic elements to be mutated, silenced, induced, or replaced has resulted in its widespread adoption in the global research community.

Integrating CRISPR genome editing with the informational power of next-generation sequencing (NGS) enables researchers to have complete control throughout their editing experiment, resulting in a better understanding of the biological systems they are modifying. NGS may be utilized at various stages of a genome editing workflow, from confirming CRISPR knockouts to analyzing off-target effects and assessing the the functional impact of gene edits.  

Gene Editing Publication Review

An overview of recent peer-reviewed gene editing research publications featuring Illumina technology

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Applications of CRISPR-Cas9 technology have been identified in the fields of basic and clinical research, therapeutics, drug development, agriculture, and the environment. Clinical research has shown potential utilization for CRISPR in such diseases as cancer, AIDS, Huntington’s disease, Duchenne muscular dystrophy, and more.

CRISPR genome editing enables researchers to create genetically modified cell lines and animal models with speed and precision. In addition to creating gene knockouts and more specific modifications, researchers can use CRISPR technology to modulate gene expression via interference (CRISPRi) or activation (CRISPRa), without altering the genomic sequence (view table).

Types of CRISPR-Enabled Genome Edits

CRISPR genome editing experiments result in mixed cell populations, with only a small subset carrying the desired edit. Researchers need to determine which cells have the desired CRISPR knockout or targeted mutation. Current methods to evaluate edits involve cleavage assays, PCR, Sanger sequencing, and NGS (view table).

NGS is the only assay that provides both qualitative and quantitative information at high resolution across the full range of modifications, meets the needs of any throughput, and can be used to monitor off-target effects.7 NGS-based targeted sequencing provides a cost-effective solution for confirming CRISPR-induced edits by focusing on regions targeted for modification.

Learn more about targeted sequencing
Methods to Check Genome Editing On-Target Efficiency

Successful implementation of CRISP/Cas9 technology should include strategies to identify and reduce off-target effects, or unintended modifications at sites other than the intended target. Computational methods to evaluate RNA specificity and predict off-target sites are commonly used during genome editing experiments.

Online tools and web-based algorithms are publicly available (view table). However, genome-wide analyses such as NGS-based whole-genome sequencing (WGS) are necessary to discover off-target sites that may escape prediction algorithms.8

Learn more about WGS
Publicly Available Analysis Tools to Predict Off-Target Effects

Genome-wide approaches for screening off-target cleavage sites include cell-based and in vitro assays. Each approach has both advantages and limitations (view table).

Unbiased Methods to Analyze
              Off-Target Effects
Single-cell RNA sequencing

Screen cell populations after CRISPR modification to determine the gene-regulatory impact of many genes in parallel in thousands of individual cells.

RNA sequencing

Assess the impact of mutations on the transcriptome as a whole or on the expression of genes/gene families.


Determine the impact of mutations on DNA-protein binding.

Methylation sequencing

Investigate the downstream impact of mutations on methylation status and chromatin remodeling.

In addition to high-resolution on- and off-target assessment and functional verification and evaluation of CRISPR knockouts and edits, NGS can be incorporated at other stages of a CRISPR genome editing workflow.

During the initial design phase, resequencing of a locus or genome (for species that lack a reference genome) can aid in RNA selection. During the process of cloning CRISPR-Cas9/guide RNA constructs, resequencing of the resulting plasmids can provide rapid and high-confidence verification of the CRISPR delivery vectors, especially for high-throughput experiments with large plasmid libraries.

How NGS Fits into a CRISPR Genome Editing Workflow
Illumina Genomics Podcast Episode 32: CRISPR-Cas9 and Beyond

Dr. Sam Sternberg, Assistant Professor of Biochemistry and Molecular Biophysics at Columbia University, discusses the biology and impact of CRISPR and genome editing.

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Genome Editing Podcast – CRISPR-Cas9 and Beyond
Agricultural Genomics
Agricultural Genomics

The simplicity and low cost of CRISPR-Cas9 technology can extend gene editing in crops from large commodity species to a wider variety of agriculturally important species.

Learn more about agrigenomics
Cancer Research
Cancer Research

The speed and simplicity of CRISPR-Cas9 technology can enable development of new cancer models and discovery of new immunotherapeutic targets and strategies.

Learn more about cancer research
Complex Disease Genomics
Complex Disease Genomics

The precision of CRISPR-Cas9 genome editing can enable development of cell and animal models of human complex diseases to investigate disease pathology.

Learn more about complex disease research
Molecular and Cellular Biology
Cellular and Molecular Biology Research

The speed, simplicity, and low cost of CRISPR-Cas9 genome editing has revolutionized cell and molecular biology in the development of gene knockouts and transgenic models.

Learn more about cellular and molecular biology research
CRISPR-Cas9: Genome Engineering Made Simple
CRISPR-Cas9: Genome Engineering Made Simple

The Illumina Scientific Affairs team discusses recent papers on applications of CRISPR-Cas9 technology.

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Targeted Resequencing and Somatic Mosaicism
Targeted Resequencing and Somatic Mosaicism

In this customer interview, a geneticist discusses using NGS in somatic mosacisim studies and possible future applications of CRISPR technology.

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