Abstract
Horizon’s
Pin-point™ base editing platform utilizes an RNA aptamer embedded within the sgRNA
to enable the recruitment of a cytidine deaminase or other effector. The
modularity of the Pin-point platform enables a high degree of flexibility,
which allows fine tuning of critical aspects of editing behaviour.
In this study, we
report the development of arrayed and pooled screening platforms to optimize
the performance of the modular Pin-point base editing system components and to
assess guide RNA functionality in a high-throughput manner. Firstly, we
describe an arrayed screening platform, demonstrating its utility in multiple
cell lines, using five different cytidine deaminases and three structurally
distinct tracrRNAs, and assess each configuration at 70 guide-specific genomic
sites. We demonstrate significant impact upon editing efficiency, editing
window size and position, and context preference with different deaminases.
Secondly, we present a flexible and adaptable pooled screening reporter
platform for high-throughput parallel assessment of >65,000 guides. We
demonstrate the ability of the pooled screening platform to detect editing in a
highly reproducible manner. Finally, we show by the use of a validation screen
that the pooled screen reporter is predictive of the editing achieved with the Pin-point
platform at the endogenous locus.
In summary, we
present the Pin-point platform as a tuneable, modular system that can be
readily adapted to address diverse editing requirements.
- Collantes
JC, Tan VM, Xu H et. al. (2021), Development and characterization of a
modular CRISPR and RNA aptamer mediated base editing system. CRISPR J,
4:58-68 (PMID 33616445)
