Tel: 1-631-275-3058 (USA)   44-208-144-6005 (Europe)       Email: info@cd-genomics.com
CD Genomics-the genomics service company
Support Documents The CD Genomics Way of Thinking Explore the scientific documents we’ve developed, including sample submission guidelines, principles, applications, and bioinformatics of genetic technologies.
Home / Resource / Support Documents / Genome Research / Next Generation Sequencing Validating Your CRISPR/Cas9 Edit

Next Generation Sequencing Validating Your CRISPR/Cas9 Edit

CRISPR/Cas9 technology

CRISPR/Cas9 technology is one of the most popular methods used for genome editing by introducing both Cas9 endonuclease and a guide RNA into the cells of interest. The guide RNA is designed to direct the Cas9 endonuclease to a particular site in the genome where it produces a double-strand break (DSB). There are generally two ways to repair the double-stranded break: non-homologous end-joining (NHEJ) or homologous directed repair (HDR). NHEJ is the main form of repair in mammalian cells. As it is error-prone, repair via the NHEJ pathway allows for insertion, deletion, loss-of-function mutations which probably result in frameshifts and affect protein expression. In addition to knockout mutations, a template DNA can be introduced to direct HDR and create mutations in the gene-of-interest. HDR faithfully copies the template sequence to the cut target site.

Next Generation Sequencing Validating Your CRISPR/Cas9 EditFigure 1. Genome editing by CRISPR/Cas9 technology is achieved via repair.

Genome edits validation

Although the CRISPR system is efficient for genome editing. However, some cells in a population will not be edited, some will have one allele edited, and some will have both alleles edited. It is important to validate genome edits after CRISPR/Cas9 experiments. Next generation sequencing (NGS), as a powerful and high-throughput approach, can be utilized for screening of CRISPR-induced mutations. NGS can simultaneously look at off-target changes in a large number of samples. When using this method, it is necessary to keep a set of control cells. Software such as CRISPResso can be used for data analysis. NGS is also suited for assessing genome edits created by ZFN (Zinc-finger nuclease) or TALEN (Transcription activator-like effector nuclease).

Sentmanat et al. (2018) described the NGS approach for genome edits validation in his published work. Briefly, CRISPR sequencing involves a two-step PCR protocol and deep sequencing. First, the target genomic site of interest is amplified with a primer that contains partial Illumina sequencing adaptors. Next, a second PCR with primers containing indices and necessary Illumina sequencing adaptors. As a result, the target regions will be amplified. The qualified PCR products are then subjected to deep sequencing with the Illumina MiSeq platform.

Detection of off-target mutations

Another limitation of CRISPR technology is the occurrence of Off-target cuts. The CRISPR system cuts not just at its target place, but also at unintended sites with similar sequences. These off-target cuts may produce undesirable and even harmful mutations. Over the past few years, scientists have established several NGS-based approaches to detect off-target mutations.

Table 1. NGS-based approaches to detect off-target mutations.

Assays Description Resources
In vitro genome-wide assays
Digenome-Seq Genomic DNA is first digested with a nuclease and then subjected to whole genome sequencing. Off-targets can be computationally identified. Kim et al. 2015
Web tool: http://www.rgenome.net/digenome-js/#!
Code: https://github.com/chizksh/digenome-toolkit2
CIRCLE-Seq Genomic DNA is sheared and circularized. The residual linear DNA is degraded. The Cas9 is then used to linearize circular DNA that contains a Cas9 cleavage site. The cleaved ends are PCR-amplified and sequenced by NGS technology to identify off-targets. Tsai et al. 2017
Code: https://github.com/tsailabSJ/circleseq
SITE-Seq Genomic DNA is cleaved using Cas9 nuclease, and Cas9 nuclease cleavage sites are biochemically tagged and enriched. High-throughput sequencing and bioinformatics analysis is then used to detect off-target cleavage sites. Cameron et al. 2017
Protocol: Protocol Exchange, doi:10.1038/protex.2017.043
Cell-based genome-wide assays
GUIDE-Seq DSBs created by the Cas9 are tagged using small double-stranded oligonucleotides, PCR amplified, and analyzed using NGS. Tsai et al. 2015
Code: https://github.com/aryeelab/guideseq
LAM-HTGTS Chromosomal translocations of off-target and on-target breaks are PCR amplified and analyzed by NGS. Frock et al. 2015
Protocol: Nat Protoc, 11:853-71, 2016
Code:http://robinmeyers.github.io/transloc_pipeline/index.html
BLISS DSBs are biochemically labeled, and their downstream sequences are PCR amplified and analyzed using NGS. Yan et al. 2017

References:

  1. Yan W X, Mirzazadeh R, Garnerone S, et al. BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nature communications, 2017, 8: 15058.
  2. Frock R L, Hu J, Meyers R M, et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nature biotechnology, 2015, 33(2): 179.
  3. Tsai S Q, Zheng Z, Nguyen N T, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nature biotechnology, 2015, 33(2): 187.
  4. Cameron P, Fuller C K, Donohoue P D, et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nature methods, 2017, 14(6): 600.
  5. Tsai S Q, Nguyen N T, Malagon-Lopez J, et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nature methods, 2017, 14(6): 607.
  6. Kim D, Bae S, Park J, et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods, 12:237-43, 2015.
  7. Sentmanat M F, Peters S T, Florian C P, et al. A survey of validation strategies for CRISPR-Cas9 editing. Scientific reports, 2018, 8(1): 888.
  8. https://www.the-scientist.com/lab-tools/new-methods-to-detect-crispr-off-target-mutations-30013
* For Research Use Only. Not for use in diagnostic procedures.
SPEAK TO OUR SCIENTISTS

What would you like to discuss?

With whom will we be speaking?

Please input "genomics" as verification code.

* is a required item.

Get cutting-edge science information from CD Genomics sent straight to your inbox every month.

SUBSCRIBE TO OUR NEWSLETTER
CONTACT CD GENOMICS

45-1 Ramsey Road, Shirley, NY 11967, USA
Tel: 1-631-275-3058 (USA)
       44-208-144-6005 (Europe)
Fax: 1-631-614-7828
Email: info@cd-genomics.com