Twisted DNA Increases CRISPR Off-target Effects

ABOVE: CRISPR-Cas9 should target specific sequences in the genome, but the conformation of DNA can make it miss the mark. © iStock, wragg

At the end of 2023, the FDA approved Casgevy for sickle cell disease, its first approval for a therapeutic that used the genome editing tool clustered regulatory interspaced palindromic repeats (CRISPR) to specifically inactivate a human gene as a treatment for a genetic disease. While CRISPR-based gene therapies could potentially treat several genetic diseases, concerns about edits at nontargeted sites have delayed its therapeutic use.

David Rueda, a single-molecule biophysicist at Imperial College London, studies off-target activity of CRISPR-associated protein (Cas) 9, one of the most widely studied CRISPR nucleases. He and his team demonstrated in a recent study published in Molecular Cell that negatively supercoiled DNA conformations increase off-target Cas9 effects.¹ These findings highlight important considerations for future applications of genome editing technology.

It's actually very similar to what you would get in Star Trek. It's a real-life tractor beam that allows us to capture and manipulate objects in space.
-David Rueda, Imperial College London

CRISPR relies on two primary components: a guide RNA (gRNA) of 17-24 nucleotides that finds the target sequence and the Cas nuclease that cuts at that precise location. Typically, for Cas9 to bind and cleave a target DNA sequence, there must be a protospacer adjacent motif (PAM) sequence immediately downstream of the target; this usually ensures on-target edits. Even so, the system is not perfect.

In a previous study, Rueda and his team demonstrated that stretching DNA increased off-target Cas9 activity.² The team next wanted to test these findings in a physiological context; they were curious to see how negative supercoiling, or under twisting of DNA that happens during normal transcription and replication, influenced off-target CRISPR Cas9 activity.

The group used single-molecule optical tweezers for their experiments. “It's actually very similar to what you would get in Star Trek,” Rueda said. “It's a real-life tractor beam that allows us to capture and manipulate objects in space.” By negatively supercoiling a linear strand of DNA isolated from a bacteriophage, the team demonstrated that this conformation greatly increased Cas9 binding to nontarget sites.³

Matt Newton, a study coauthor who is currently a visiting scientist at The Francis Crick Institute, was excited to see these effects. “I then wanted to see, would this actually change anything if you were targeting actual human genomic DNA?” he said.

The team chose an in vitro screening method, circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), for their human genomic DNA tests.⁴ The researchers fragmented and circularized DNA from a human cell line and treated it with Cas9 and DNA gyrase to induce negative supercoiling in vitro. Cas9 activity linearized the DNA circles, and the team sequenced the linear fragments, revealing the sequences where the enzyme cut. The team showed that DNA gyrase treatment more than doubled the number of off-target cleavage sites.

The next step was to validate their results in human cells. The team treated cells with Cas9 and used INDUCE-seq, a method that enabled them to selectively sequence fragments with double-stranded DNA breaks, a hallmark of Cas9 cleavage.⁵ When the researchers compared the off-target sequences from INDUCE-seq sequences with those from their CIRCLE-seq experiments, they found less than 25 percent alignment between the results. These data suggested additional effects from DNA structure being altered by other cellular means, such as transcription. On testing their hypothesis, the team found that more than 70 percent of the off-target sites mapped to regions with high transcriptional activity.

Schematic of Cas9 activity affected by sequence mismatching and DNA conformation.

(1) The guide RNA (gRNA) and PAM region typically direct the Cas9 protein to its specific target sequence for DNA cleavage (top panel). (2) Mismatches (highlighted in orange) between the DNA and gRNA prevent binding and cleavage (middle panel). (3) Altered DNA conformations may permit Cas9 binding with imperfect sequence alignment (bottom panel).

“The effects of DNA supercoiling on the genome-wide activity of Cas9 are relatively interesting and novel,” said Shengdar Tsai, a molecular geneticist at St. Jude Children’s Research Hospital who was not involved in the study but who holds patents for developing genome sequencing technologies, including CIRCLE-seq. He is interested in seeing additional studies exploring the frequencies of these off-target sites in cells and their effects through deeper profiling. “Understanding the genome-wide activity of editors is a really important question, especially towards therapeutic applications,” Tsai said. “We really want to know what these editors are doing in the genomes of living cells.”

“[These findings] can provide an additional mechanism by which off-target cuts are being recruited in cells and can perhaps help us design new CRISPR-Cas systems that will be more accurate,” Rueda said.