Researchers Detect DNA Modifications Created by Gene Editing

Method finds, measures unintended DNA changes after CRISPR-Cas9 therapy

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by Steve Bryson, PhD |

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This is an illustration of a DNA strand.

Researchers have developed a method to detect and measure large unintended DNA modifications created by CRISPR-Cas9 gene-editing methods currently being investigated as treatments for sickle cell disease (SCD).

High levels of unintended gene modifications that occurred at selected target sites demonstrate the need for a more careful evaluation of gene-editing outcomes, the researchers noted.

Gang Bao, PhD, a bioengineer at Rice University in Texas, began the work after studies suggested the presence of large DNA changes due to gene-editing methods. “That’s when we started looking into what we can do to quantify them, due to CRISPR-Cas9 systems designed for treating sickle cell disease,” Bao said in a press release.

The study, “Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing,” was published in the journal Science Advances.

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CRISPR-Cas9 is a gene-editing tool currently being investigated as a method to correct SCD-causing mutations in the HBB gene. This gene provides instructions for making a component of hemoglobin — the protein that transports oxygen in red blood cells.

The treatment approach involves collecting hematopoietic stem cells (HSC), which give rise to red blood cells, from an SCD patient, then modifying them with CRISPR-Cas9 before returning the corrected stem cells to the same patient in the form of a stem cell transplant.

CRISPR uses an RNA guide (gRNA) to target a specific location on the DNA strand, and then the attached Cas9 enzyme cleaves both double-helix strands at that location, called double-stranded breaks (DSBs).

Target genes are then altered by a natural DNA repair process that results in small insertions and deletions of DNA, called INDELs, at the Cas9 cut sites. INDELs are typically smaller than 50 base pairs (bp) — the fundamental building blocks of double-stranded DNA. Current methods, such as PCR, can accurately measure the occurrence and impact of INDELs.

Almost all gene-editing methods require highly efficient cutting at the selected target sites (on-target). Still, cuts to DNA also can occur off-target in places other than the chosen target site, leading to potential side effects. As a result, off-target sites have been studied extensively, and approaches have been developed to reduce off-target effects.

Limited measurement methods

Recent studies indicated that, in addition to INDELs, Cas9 cutting might introduce large DNA deletions (more than 200 bp) and large DNA insertions (50 bp or more) at on-target sites. However, current methods to accurately measure these on-target modifications, including PCR, are limited.

Bao and his team have now developed a method to measure large changes in DNA at on-target sites that occur due to CRISPR-Cas9 gene editing.

“We do not have a good understanding of why a few thousand bases of DNA at the Cas9 cut site can go missing and the DNA double-strand breaks can still be rejoined efficiently. That’s the first question, and we have some hypotheses,” said Bao. “The second is, what are the biological consequences? Large deletions (LDs) can reach to nearby genes and disrupt the expression of both the target gene and the nearby genes. It is unclear if LDs could result in the expression of truncated proteins.”

“You could also have proteins that misfold, or proteins with an extra domain because of large insertions,” Bao added. “All kinds of things could happen, and the cells could die or have abnormal functions.”

Their approach, called LongAmp-seq, combined two techniques: single-molecule real-time (SMRT) sequencing with dual unique molecular identifiers (UMI). Computer-based methods, or bioinformatics, then analyzed data.

“To quantify large gene modifications, we need to perform long-range PCR, but that could induce artifacts during DNA amplification,” Bao said. “So we used UMIs of 18 bases as a kind of barcode.”

“We add them to the DNA molecules we want to amplify to identify specific DNA molecules as a way to reduce or eliminate artifacts due to long-range PCR,” Bao added. “We also developed a bioinformatics pipeline to analyze SMRT sequencing data and quantified the LDs and large insertions.”

The team first analyzed CRISPR-Cas9 gene edits (induced by the R-66S gRNA) applied to the HBB gene in HUDEP2 cells, an hematopoietic stem and progenitor cell (HSPC)-derived blood cell precursor cell line. Results demonstrated 35.4% of large deletions (200 bp or more), 5.8% of intermediate DNA deletions (50 to 200 bp), and 1.9% large insertions (50 bp or more), alongside 52.8% INDELs.

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Next, both small and large gene modifications were measured in SCD HSPCs induced by three other gRNAs — R-02, SD-02, and BCL11A — all designed to treat SCD. R-02 generated a double-stranded break on the HBB gene close to the sickle mutation site, SD-02 introduced a deletion to activate fetal hemoglobin, and BCL11A gRNA created a double-stranded break, also to induce fetal hemoglobin production.

R-02 induced 11.7% large deletions, 6.1% intermediate deletions, and 1.1% of large insertions, while SD-02 triggered 14.3% large deletions, 2.65 intermediate deletions, and 1.05% of large insertions. Gene modifications due to BCL11A included 13% large deletions, 4.7% intermediate deletions, and 1.5% large insertions. Similar findings were observed after editing immune T-cells to demonstrate the creation of large deletions in primary cells.

“Our study provided a comprehensive analysis of gene editing outcomes by five Cas9/gRNA [edits] in cell lines, HSPCs, and T cells,” the researchers wrote. This “revealed high levels of unintended gene modifications; and demonstrated the need for more careful evaluation of gene editing outcomes, especially for therapeutic genome editing using CRISPR-Cas9.”

Bao added that they are currently looking downstream to analyze the consequences of long deletions on protein production. “Then we’ll move on to the protein level,” Bao said. “We want to know if these large deletions and insertions persist after the gene-edited HSPCs are [transplanted] into mice and patients.”