The role of long-read sequencing in validating CRISPR outcomes


Neil Ward, general manager of PacBio EMEA 

With applications of CRISPR across the spectrum of microbial, plant and gene therapy increasingly being realised, enthusiasm from the research community for this technology is not expected to wane. It has the potential to, among other applications, create knock-out animal models for use in research, revolutionise agriculture and even contribute to the development of CAR-T therapies for cancer. 

But as with any nascent technology, checks and balances are critical. It is important to ensure that CRISPR applications result in only the intended genomic edits. Researchers have long struggled with more genomic changes than expected taking place during experiments – including deletions of stretches of DNA. And when making changes to DNA, specificity is essential. 

Although recent advances have been made, with the SpCAS9 variant, SpRY offering more precision, accurate validation is still vital. For the true potential of CRISPR to be realised and made scalable, researchers need to understand the complete scope of genomic changes that occur whatever variant is employed.

It is essential that we build a strong foundation for CRISPR experimentation to be used in valid, safe, and repeatable ways. 

Avoiding unintended mutations

One approach to building this foundation lies in the use of accurate, long-read genomic sequencing technology. By employing long-read sequencing to characterise the effects of a CRISPR experiment, results can be validated, and unintended off-target mutations can be identified. This gives researchers the confidence to move their experiments on to the next stage without unexpected mutations leading to failure or inconsistencies. 

A 2018 study from researchers at the Wellcome Sanger Institute demonstrated why visibility into the full picture of genomic changes triggered by gene editing is critical to progressing research projects. The Sanger team used long-read sequencing to identify substantial mutagenesis after use of CRISPR – including entire sections of the genome being rearranged or lost completely.

The team re-ran their experiments to try and gain a clearer picture of which changes CRISPR was causing, with varying results on each occasion. After repeating the same experiment four times, they found no consistency in outcomes, saying, “Each biological replicate differed substantially, despite a large number of unique deletion events sampled, indicating that the diversity of potential deletion outcomes is vast.”

Identifying mutations that occur adjacent to target sites is standard practice in CRISPR validation, and short-read sequencing is often used to achieve this. However, as the Sanger team found, further mutations also occur many kilobases up- and down-stream from these loci that are difficult if not impossible to spot without long-read sequencing. Deletions and rearrangements that take place away from the target site can still have a significant impact, with long deletions impacting binding sites and other important regulatory elements of the protein. 

Getting the right answers

With a greater understanding of the scale of the changes caused by CRISPR editing techniques, we can more reliably measure the outcomes of the method. Post-experimental assessments must give an accurate and comprehensive view of the genome, and not rely on using solely short-read sequencing in CRISPR validation. 

CRISPR holds great promise in the future of healthcare and in propelling research breakthroughs. But to maintain confidence and enable CRISPR to achieve its potential, we must continue to prioritise the validation and comprehensive characterisation of any such editing experiments.

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