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Global Translational Medicine ABE gene therapy for CVDs

Table 2. Representative methods to determine genomic off‑target effects
Classification Name Advantage Disadvantage
Computational Cas-OFFinder [67] Simple, cheap and expedient Biased toward sgRNA- dependent off-target sites. Still
prediction CasOT [68] require further experimental validation.
DeepCRISPR [69]
Elevation [70]
In vitro detection on Digenome-seq [73] Unbiased and sensitive Only applicable in vitro. With false positive and false
purified genomic DNA CIRCLE-seq [74] negative hits due to the lack of intracellular factors.
SITE-seq [72]
EndoV-seq [71] Specifically designed for ABE
Label and capture in cell IDLV capture [76] Unbiased, sensitive and in living cells Only applicable in cell culture. With false positive and false
culture GUIDE-seq [75] negative hits due to the lack of physiological relevance.
Direct detection in WGS [77] Unbiased and suitable for clinical samples Expensive and relatively low sensitivity
animal tissues DISCOVER-seq [78] Unbiased and suitable for clinical samples Relatively low sensitivity
GUIDE-tag [79] Unbiased and in animal tissues Technically complicated and not suitable for clinical samples
Digenome-seq: In vitro Cas9-digested whole-genome sequencing, CIRCLE-seq: Circularization for reporting of cleavage effects by sequencing,
SITE-seq: Selective enrichment and identification of adapter-tagged DNA ends by sequencing, IDLV: Integrase-deficient lentivirus,
GUIDE-seq: Genome-wide, unbiased identification of DSBs enabled by sequencing, WGS: Whole genome sequencing. DISCOVER-seq: Discovery of
in situ Cas off-targets and verification by sequencing

and accurate enough to evaluate ABE-induced off-target editing tools such as ABE, therapeutic genome editing in
effects in gene therapy remain to be determined. human bodies has entered clinical trials and will likely become
So far, two major approaches have been applied to reduce a reality in near future. In this review, we showcase the power
the off-target effects by ABE. The first method harnesses of ABE in CVD therapy and recommend more cardiovascular
protein structure information or directed evolution researchers to embrace ABE as a new weapon to tackle CVDs.
technology to engineer more accurate ABE mutants. For Acknowledgments
example, arginine 153 (R153) within TadA was reported
to mediate its RNA editing activity; thus, R153 deletion None.
was implemented in ABEs to minimize its RNA off-target
effects . Importantly, the recently established ABE9 also Funding
[81]
drastically reduced both RNA off-target effects and Cas9- This work was funded by the National Key R&D Program
independent DNA off-target effects, in addition to the of China (2022YFA1104800), the National Natural
aforementioned impact on bystander effects . Science Foundation of China (82222006, 32100660,
[59]
Another plausible method to reduce the off-target effect 82170367, 82100349 and 82200405), Beijing Nova
works by controlling the duration of ABE expression. Program (Z211100002121003 and 20220484205), and the
Because RNA exhibits a high turnover rate, RNA off- Postdoctoral Science Foundation of China (2021M692253).
target effects will gradually taper off once the ABE stops Conflict of interest
expressing. Similarly, once the on-target DNA editing
is accomplished, ABE activities should be terminated No potential conflicts of interest were disclosed.
to avoid further accumulation of off-target edits in the
genome. Based on this rationale, LNP vectors are more Author contributions
suitable than AAV vectors in delivering ABEs to the liver, Conceptualization: Zihao Tao, Yuxuan Guo, Fei Gao
as LNP-mediated ABE expression only persists for days, Writing – original draft: Luzi Yang, Zihao Tao, Xiaoteng
while AAV-mediated gene expression can last for years. Ma, Xuanhui Zhang, Yuxuan Guo
Writing – review & editing: Luzi Yang, Zihao Tao, Xiaoteng
5. Conclusions Ma, Yuxuan Guo, Fei Gao
CRISPR/Cas9-based genome editing has revolutionized
biomedical research, including cardiovascular research, in the Ethics approval and consent to participate
past decade. With the emergence of more advanced genome Not applicable.

Volume 2 Issue 1 (2023) 9 https://doi.org/10.36922/gtm.232