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.

Figure 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.

References:

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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.
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8. https://www.the-scientist.com/lab-tools/new-methods-to-detect-crispr-off-target-mutations-30013