How many is one too many? Off target mutations in CRISPR reported by whole genome sequencing


For the past few years, CRISPR has constantly been in the spotlight for a myriad of reasons. In addition to the rapid advancements in the technology and its expanding applications, umpteen number of news stories have covered the battle for the patent between competing groups. Whether we talk about benign mosquitoes that do not transmit malaria or extraordinarily muscular beagles or mini pigs, CRISPR is everywhere and everyone is using it.

CRISPR is a natural adaptation found in bacteria which allows it to defend itself from invading bacteriophages. The host bacteria creates a memory of the invading bacteriophage DNA sequence in its own genome. This serves as a memory that can be used to cleave specific DNA sequences of invading organisms by the cleavage enzyme Cas9 in the event of an infection. Scientists have been able to use this ability of the Cas9 enzyme to undertake targeted gene editing in in vitro as well as in vivo model systems.

Last year backed by the venture capitalist Sean Parker, The National Institute of Health approved a proposal for human clinical trials of CRISPR. Taking it one step ahead, Chinese oncologists at Sichuan University injected CRISPR-Cas9 modified cells in a patient suffering from an aggressive form of lung cancer in October last year. Despite the ease of use offered by CRISPR to completely ablate gene expression, there have been concerns regarding the specificity and efficiency in practice.

Are we about to open Pandora’s box?

A recent study revealing several unexpected mutations after CRISPR-Cas9 editing in vivo has set alarm bells ringing.

A team of researchers in the US set out to repair a genetic mutation known to cause blindness in mice. Using CRISPR gene editing, they were able to successfully correct the targeted mutation in each of the two mice they treated. In a later study, the team sequenced the entire genome of two mice that had undergone CRISPR gene editing, and one healthy control. They observed an alarming number of additional DNA changes — more than 1,600 per mouse — in areas of the genome they did not intend to modify. Researchers in the past used computer algorithms to identify and examine areas most likely to be affected by off-target mutations.

“These predictive algorithms seem to do a good job when CRISPR is performed in cells or tissues in a dish, but whole genome sequencing has not been employed to look for all off-target effects in living animals”  Alexander Bassuk, team member, University of Iowa.

“Researchers who aren’t using whole genome sequencing to find off-target effects may be missing potentially important mutations, even a single nucleotide change can have a huge impact.”- Dr. Tsang

What does this mean for regulatory bodies?

This new piece of information from Schaefer et al comes at a crucial moment in time as regulatory bodies across the world are getting ready to approve CRISPR-based therapies. Whole genome sequencing could be an important emerging benchmark for approved CRISPR therapies.