Accelerate CRISPR genome editing using next-generation sequencing

NGS enables researchers to have 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 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 Research Review
Gene Editing Publication Review

See summaries of recent peer-reviewed gene editing research publications featuring Illumina NGS 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 sickle cell disease, 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 gene knock-ins, 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) may be 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 NGS approaches for detecting off-target cleavage sites include cell-based assays (in live or fixed cells) as well in vitro assays. Each method has both advantages and limitations (view table).

Unbiased Methods to Analyze
              Off-Target Effects

Featured Genome Editing Content

Genomics podcast
CRISPR-Cas9 and Beyond - Illumina Genomics Podcast Episode 32 

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

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CRISPR-Cas9 Genome Engineering
CRISPR-Cas9: Genome Engineering Made Simple

The Illumina Scientific Affairs team summarizes key publications on applications of CRISPR-Cas9 technology.

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Genetics and the Agriculture Industry
Facing the Future: How Genetics Will Continue to Transform the Agriculture Industry

Genotyping will enable dairy farms to adapt to changing climate and farming conditions, while producing milk that needs less processing.

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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.

Featured Products

NextSeq 2000 System
NextSeq 2000 System

Groundbreaking benchtop sequencers allow you to explore new discoveries across a variety of current and emerging applications, with higher efficiency and fewer restraints.

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TruSeq Stranded Total RNA
TruSeq Stranded Total RNA

A robust, highly scalable whole-transcriptome analysis (RNA-Seq) solution for a variety of species and sample types, including human, mouse, and formalin-fixed, paraffin-embedded (FFPE) tissue.

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TruSeq ChIP Library Preparation Kit
TruSeq ChIP Library Preparation Kit

Simple, cost-effective chromatin immunoprecipitation sequencing (ChIP-Seq) DNA library preparation, with master mixes and robust multiplex capabilities.

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TruSeq Methyl Capture EPIC Library Prep Kit
TruSeq Methyl Capture EPIC Library Prep Kit

This kit combines next-generation sequencing with epigenetic insights to accelerate biomarker discovery and understand methylation’s role in gene regulation.

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In addition to high resolution on- and off-target assessment and functional analysis of CRISPR edits, NGS can be incorporated at additional stages of the 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
Francis deSouza on Genomics, CRISPR, and the Future

Francis deSouza, President and CEO of Illumina, Inc., took the stage at Aspen Ideas: Health in Colorado to educate the audience on the impact genomics is having across healthcare, and the urgency in bringing its benefits to more people, more quickly.

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CEO Francis deSouza Speaks at Aspen Ideas: Health
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 gene editing has revolutionized cell and molecular biology in the development of gene knockouts and transgenic models.

Learn more about cellular and molecular biology research
NGS Technology
NGS Technology

Ultra-high throughput, scalability, and speed enable biological understanding never before possible.

Explore NGS
Publication Reviews
Publication Reviews

These summaries of peer-reviewed publications highlight how Illumina technology is furthering scientific research.

Read Reviews
  1. Cong L, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819-823.
  2. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823-826.
  3. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015;33:538-542.
  4. Chu VT, Weber T, Wefers B, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015;33:543-548.
  5. Qi LS, Larson MH, Gilbert LA, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152:1173-1183.
  6. Cheng AW, Wang H, Yang H, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23:1163-1171.
  7. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281-2308.
  8. Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet. 2016;17:300-312.
  9. Gabriel R, Lombardo A, Arens A, et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol. 2011;29:816-823.
  10. Tsai SQ, Zheng Z, Nguyen NT, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187-197.
  11. Chiarle R, Zhang Y, Frock RL, et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell. 2011;147:107-119.
  12. Crosetto N, Mitra A, Silva MJ, et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods. 2013;10:361-365.
  13. Kim D, Kim S, Kim S, Park J, Kim JS. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. 2016;26:406-415.
  14. Tsai SQ, Nguyen NT, Malagon-Lopez J, Topkar VV, Aryee MJ, Joung JK. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods. 2017;14:607-614.