Science Advances: Even good gene editing can go bad

A lab at Rice University is working to uncover potential threats to the effectiveness and safety of treatments based on CRISPR-Cas9, a Nobel laureate) gene-editing technology, even as the technology appears to be working as
planned.

Rice University bioengineer Gang Bao and his team note in a paper published in Science Advances that while off-target editing of DNA has long been a concern, the invisible changes that accompany off-target editing also need to be recognized — and quantified
.

He noted that a 2018 paper in Nature Biotechnology showed a large number of gaps
.
"That's when we started looking at how to quantify them because of the CRISPR-Cas9 system
for treating sickle cell disease," he said.

Gang Bao has been conducting research on CRISPR-Cas9 as a tool to treat sickle cell disease, an exploration that brings him and his colleagues closer
to a cure for sickle cell disease.
Now, researchers are concerned that large deletions or other undetected changes due to gene editing may persist during stem cell division and differentiation, with long-term health effects
.

Professor Bao said: "We do not yet have a good understanding of why the thousands of base DNA at the Cas9 cleavage site are lost, while DNA double-strand breaks can still be efficiently reattached
.
This is the first question, we have some assumptions
.
The second question is, what are the biological consequences? Large deletions (LDs) can reach neighboring genes and disrupt the expression
of the gene of interest and neighboring genes.
It is unclear whether LD causes truncation of protein expression
.
“

"There may also be protein misfolding, or proteins having extra domains
due to a large number of insertions," he said.
All sorts of things can happen, and cells can die or function abnormally
.
”

His lab has developed a procedure that uses single-molecule real-time (SMRT) sequencing with dual unique molecular identifiers (UMIs) to discover and quantify unintentional LDs, as well as large insertions and local chromosomal rearrangements (with small insertions/deletions (INDELs))
at Cas9 target cut sites.

Professor Bao said: "To quantify large genetic modifications, we need to perform remote PCR, but this can cause artificial interference
during DNA amplification.
So we used an 18-base UI as a kind of barcode
.
”

"We add them to the DNA molecules we want to amplify to identify specific DNA molecules, which is a way to
reduce or eliminate the artifacts produced by remote PCR," he said.
We also developed a bioinformatics pipeline to analyze SMRT sequencing data and quantify
LD and large insertions.
”

Bao's lab's tool, called LongAmp-seq (for long amplicon sequencing), can precisely quantify small INDELs and large lds
.
Unlike SMRT-seq, which requires the use of a long-read sequencer normally only available in core facilities, LongAmp-seq can be performed
using a short-read sequencer.

To test this strategy, a team of labs led by Rice alumnus Julie Park used Streptococcus pyogenic Cas9 to edit β-globin (HBB), γ-globin (HBG), and B-cell lymphoma/leukemia 11A (BCL11A) enhancers in hematopoietic stem and progenitor cells (HSPCs) from sickle cell disease patients, as well as the PD-1 gene
in primary T cells.

They found that large deletions of up to several thousand bases occur frequently in HSPCs: up to 35.
4 percent in HBB genes, up to 14.
3 percent in HBG genes, up to 15.
2 percent in BCL11A genes, and up to 15.
2 percent
in the PD-1 gene in T cells.

Because two specific CRISPR guides tested by Bao's lab are being used in clinical trials to treat sickle cell disease, he said, it's important to
determine the biological consequences of large genetic modifications caused by cas9-induced double-strand breaks.

Professor Bao said the Rice team was currently analysing downstream the consequences of prolonged deletion of messenger RNA, a medium
that carries ribosome codes to make proteins.
"Then we will continue to study protein levels, and we want to know if these large-scale deletions and insertions will continue
after gene-edited HSPCs are transplanted into mice and patients.
"