Cas9-Mediated Random Access in DNA Data Storage

Synthetic DNA is an excellent medium for long-term digital data storage as it can encode data with high density and durability. One requirement of practical DNA data storage systems is the ability to selectively access data files (a process known as "random access") to lessen decoding latency and cost. We and others have previously explored PCR-addressable access within DNA data pools. However, this process is not ideal due to the additional sample prep, long cycle times, and occurrence of artifacts involved with PCR amplification. Here, we demonstrate a new random access approach in which files can be selected in multiplex using a CRISPR-Cas9 target address and then decoded using a nanopore sequencer. We designed and validated this random access architecture within a pool of 1.6 million DNA sequences representing 25 individual data files. This strategy has several advantages over previous work, including 1) decreased time-to-decoding, 2) increased one-pot multiplexability, and 3) increased energy efficiency due to replacing thermocycling steps with an isothermal reaction.