'Bystander' Cs meet their match in gene-editing technique
Biomolecular engineers' tool sets precisely fix cytosine mutations in
human genome

Date:
July 15, 2020
Source:
Rice University
Summary:
Biomolecular engineers have developed new tools to increase the
accuracy of CRISPR single-base editing to treat genetic diseases.



FULL STORY ========================================================================== Biomolecular engineers at Rice University have found a C-worthy technique
that dramatically enhances the accuracy of gene editing.


==========================================================================
The Rice lab of biomolecular engineer Xue Sherry Gao has introduced a
set of tools that increase the accuracy of CRISPR-based edits in disease sequence models up to 6,000-fold compared with a current base editor,
BE4max, that is considered state-of-the-art.

The work appears in the open-access journal Science Advances.

Cytosine base editors are able to convert cytosines (C) to thymines
(T) in the human genome, which consists of three billion Cs, Ts, As
(adenine) and Gs (guanine). The base pairs of C-G and A-T encode the
genetic information in DNA.

Even one incorrect base in the human genome -- a mutation -- can lead
to genetic diseases.

"T-to-C mutations called single nucleotide polymorphisms account for
somewhere around 38% of human pathogenic diseases," Gao said. "Cytosine
base editors provide great promise to potentially treat these diseases
by reversing the C mutation back to T.

"However, when there is a 'bystander' C located right upstream of the
targeted C, the previous technology could not distinguish between the
Cs, and both would be changed to Ts," she said. "We really only want
to correct the disease- relevant C to a T and leave the bystander
C unmodified.



========================================================================== "That provided the motivation for this project," Gao said. "We want to
engineer a new cytosine base editor that can precisely modify the single targeted C while minimizing the unwanted C editing when consecutive 'CCs'
are positioned in the editing window." The Gao lab seeks to develop
base editors through a series of protein- engineering efforts. The
new cytosine base editors, called A3G-BEs, have dramatically increased precision by only editing the second of consecutive Cs.

To put their tests in "disease-relevant contexts," the Gao lab used their
tools to modify human cells to create cystic fibrosis and several other
disease model cell lines. All showed significant success at precisely
creating the desired pathogenic C-to-T mutation, particularly the cystic fibrosis cells, which all three of the A3G-BE variants perfectly modified
more than 50% of the time compared to 0.6% for BE4max.

The Gao lab also tested its new A3G-BEs' potential to correct mutations in disease treatment applications, including cystic fibrosis, holocarboxylase synthetase deficiency and pyropoikilocytosis, a type of anemia.

In experiments on cell models containing pathogenetic mutations,
A3G-BEs significantly outperformed BE4max. In the case of holocarboxylase synthetase deficiency, the editor perfectly corrected only the target C nucleotides in more than 50% of the sequences, with a 6,496-fold higher correction than BE4max.

"We also identified 540 human pathogenic single nucleotide polymorphisms
that could be precisely correctable by our A3G-BEs," Gao said. "A3G-BE
also appears to decrease off-target edits (unwanted edits to other parts
of the genome that could introduce mutations) at both the DNA and RNA
levels." Decreasing off- targets has been a prime goal of CRISPR research.

"There are three billion base pairs in humans," she said. "I believe this technology's level of precision is going to be a significant contributor
toward treating genetic disease."

========================================================================== Story Source: Materials provided by Rice_University. Note: Content may
be edited for style and length.


========================================================================== Journal Reference:
1. Sangsin Lee, Ning Ding, Yidi Sun, Tanglong Yuan, Jing Li, Qichen
Yuan,
Lizhong Liu, Jie Yang, Qian Wang, Anatoly B. Kolomeisky, Isaac
B. Hilton, Erwei Zuo, Xue Gao. Single C-to-T substitution using
engineered APOBEC3G- nCas9 base editors with minimum genome- and
transcriptome-wide off-target effects. Science Advances, 2020; 6
(29): eaba1773 DOI: 10.1126/ sciadv.aba1773 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200715154244.htm

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