Emerging applications for DNA writers and molecular recorders

Abstract

Natural life is encoded by evolvable, DNA-based memory. Recent advances in dynamic genome-engineering technologies, which we collectively refer to as in vivo DNA writing, have opened new avenues for investigating and engineering biology. This Review surveys these technological advances, outlines their prospects and emerging applications, and discusses the features and current limitations of these technologies for building various genetic circuits for processing and recording information in living cells.

Fig. 1.. DNA-writing technologies and their emerging applications.

(A) A schematic representation of a DNA writer (left) and mutation signatures generated by precise (middle) and pseudorandom (right) DNA writers. S0 and S1 indicate unmodified (memory state 0) and mutated (memory state 1) alleles, respectively. B1 to B5 indicate random memory states 1 to 5 that are generated by pseudorandom DNA writers and could serve as distinct barcodes. (B) Schematic representation of a molecular recorder and strategies that can be used to couple its activity to signals of interest (left), along with examples of applications in basic research (middle) and biotechnology (right). (C) Examples of evolutionary cellular engineering applications enabled by DNA writers. (D) Various forms of logic and computation can be achieved by layering multiple precise recorders. (E) Examples of strategies for high-throughput mapping of interactions or activities of variant libraries by DNA writers. (F) Pseudorandom DNA writers can be used to develop dynamic in vivo genetic barcoding schemes that distinctively and progressively mark cellular lineages over time.

Fig. 2.. Precise DNA-writing technologies.

(A) Site-specific recombinases. (B) Recombineering. (C) Base editing. CDA, cytidine deaminase; d/nCas9, dCas9 or nickase Cas9. (D) Digital versus analog recording (see Box 1).

Fig. 3.. Pseudorandom DNA-writing technologies.

(A) A schematic representation of the Cas9 nuclease-based DNA-writing system used for lineage tracing in zebrafish in (11). S[R1], S[R2], and S[Rn] represent random memory states 1, 2,…, n generated by the pseudorandom DNA writers. These random memory states individually, or in combination with other random memory states, can serve as distinct barcodes (e.g., B1 = S[R1] and B2 = S[R2][R1]). (B) A schematic illustration of the Cas9 + stgRNA recording system. (C) DNA writing by the Cas1-Cas2 spacer acquisition system. (D) The strategy used in (3) to record temporal information into a CRISPR array.