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Global Translational Medicine ABE gene therapy for CVDs

double-stranded break (DDB), which can be repaired DNA . Dr. David Liu’s team solved this problem through
[9]
through either non-homologous end jointing (NHEJ) or a directed evolution experiment on TadA, uncovering
homology-directed recombination (HDR) . mutations at or near the TadA D108 residue as the key
[6]
[9]
HDR-based genome editing can precisely write DNA modifications to enable ABE activity on DNA substrates .
sequences at the will of the scientists by providing a template Additional molecular evolutions and trials-and-errors in
DNA donor. However, the application of HDR is limited testing mutation combinations in TadA eventually lead
by its low editing efficiency, which is further complicated to the TadA7.10 mutant as well as the corresponding ABE
[9]
by the small nucleotide insertions and deletions (Indels) prototype called ABE7.10 .
that are simultaneously created by NHEJ reaction . Indels Because TadA and Cas9 were derived from prokaryotes,
[5]
usually outnumber the HDR products among the genome- ABE7.10 requires additional modifications to adapt to
edited cell population, depositing unwanted frame-shifting the applications in mammalian cells. First, the codon
mutations, so conventional CRISPR/Cas9 genome editing usage in the ABE7.10 gene was modified by introducing
is usually more useful in gene silencing applications, while silent mutations so that ABE7.10 was better translated
more efficient and precise genome editing tools, such as into proteins by tRNAs preferentially expressed in
the base editors (BEs), are necessary for the correction of mammals. The second adaption involved the addition of
disease-causing genetic variants. nuclear localization signals (NLSs), so ABE7.10 could be
BEs are initially derived from the Cas9 nickase (nCas9) , transported into the cell nucleus for genome editing in
[8]
which is a Cas9 mutant that only cuts one DNA strand, eukaryotes. After the initial codon optimization and NLS
[13]
greatly reducing DDB formation and the introduction of incorporation efforts in ABE7.10 , these parameters
indels at the edited loci. BEs are constructed by fusing were, further, optimized in following studies, eventually
[13,14]
nCas9 with an engineered deaminase that preferentially leading to a more robust ABE variant called ABEmax .
catalyzes nucleotide conversions on DNA [8,9] . The two most The wildtype TadA protein acts as a dimer. Unlike in
[8]
widely used BEs are the cytosine base editors (CBEs) and bacteria cells, where the TadA in ABEs can pair with the
the adenine base editors (ABEs) . In CBEs, nCas9 is endogenous TadA to facilitate genome editing, mammalian
[9]
fused to the cytidine deaminase APOBEC1 to catalyze the cells do not express TadA. To solve this problem, in ABE7.10
nucleotide conversion of cytidine (C) to uracil (U), which is and ABEmax, an evolved TadA was fused in tandem with
next modified as a thymidine (T) by the endogenous DNA a wildtype TadA to allow intramolecular dimerization .
[9]
repair system. In ABEs, the tRNA adenosine deaminase However, in following studies, additional molecular
(TadA) is engineered to enable adenosine (A) deamination evolution led to the discovery of ABE8e and ABE8.20, in
into inosine (I), which is next converted to guanidine (G) which the TadA mutant can function as a monomer and
(Figure 1). exhibit even higher editing activity [15,16] (Table 1).
Among the SNVs that are known to associate with 2.2. The scope of the editable adenines
human diseases, nearly half of them are mutated from
the C·G pair to the T·A pair [9,10] , falling into the situation Several factors determine if a specific adenosine is suitable
in principle reversible by ABEs. ABEs also demonstrate for ABE. First, this nucleotide must be positioned within
simpler domain structures and lower off-target effects an “editing window” defined by the position of the sgRNA.
as compared to CBEs [11,12] (Figure 1). Thus, ABEs have In the CRISPR/Cas system, sgRNAs must be placed next to
become the most popular BE tools that are promising in a protospacer adjacent motif (PAM), which is determined
translational medicine. by the intrinsic property of the Cas protein. For example,
the PAM sequence of a wildtype SpCas9 protein is NGG
In this review, we navigate our focus on the key technical (N means any nucleotide) [17,18] , while natural SaCas9 uses
features of ABEs and introduce the recent landmark gene NNGRRT (R means A or G) as the PAM sequence . The
[19]
therapy studies for CVDs. We also discuss about the PAM sequence determines the location, where the CRISPR/
technical concerns on the road toward successful clinical Cas9 system unwinds the DNA to form R-loop , which
[20]
applications. exposes the single-strand DNA in the editing window to
2. Basic parameters for ABE therapy TadA for the deamination reaction (Figure 1).
In addition to PAM, the editing window of ABEs is also
2.1. Editing efficiency
determined by their enzyme activity and their structural
The effort to fuse TadA to nCas9 to edit adenosine features. Usually, the editing windows of ABEs are 4-5nt
initially failed with no detectable edits , because natural wide. With the use of more robust TadA variants, the
[9]
adenosine deaminases usually function on RNA but not editing windows of ABEs can be broadened [15,16] . However,

Volume 2 Issue 1 (2023) 2 https://doi.org/10.36922/gtm.232