Single-Nucleotide-Resolution Computing and Memory in Living Cells

Abstract

to encode a wide range of order-independent, sequential, and temporal logic and memory 26 operations. Furthermore, we show that these operators can be used to perform both digital and 27 analog computation, and record signaling dynamics and cellular states in a long-term, autonomous, 28 and minimally disruptive fashion. Finally, we show that the platform can be functionalized with The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/263657 doi: bioRxiv preprint first posted online Feb. 15, 2018; 3 Main Text: 40 Robust and scalable molecular recording and computation platforms in living cells are key to 41 enabling a broad range of bioengineering and biomedical applications. Unlike their silicon-based 42 counterparts that have access to large capacities of addressable memory registers, synthetic genetic 43 circuits currently have very limited information storage capacities and existing methods for 44 encoding information into cellular memory, as well as strategies for integrating such memory with 45 logic operations, are challenging to scale. 46 Genomic DNA is an ideal medium for biological memory since it is ubiquitously present, 47 naturally replicated at high fidelity within cells, and compatible with natural biological operations. 48 In recent years, several strategies for encoding biological information into DNA and integrating 49 these memories with cellular computers have been described (Farzadfard and Lu, 2014; Kalhor et 50 al., 2017; McKenna et al., 2016; Perli et al., 2016; Roquet et al., 2016; Siuti et al., 2013). However, 51 these methods remain limited in their encoding capacity and scalability. For example, site-specific 52 recombinases that flip or excise targeted DNA segments have been used to create digital memory, 53 sequential logic, and biological state machines in living cells (Roquet et al., 2016; Siuti et al., 54 2013). However, a different recombinase is required for every unique event that one wishes to 55 record, thus limiting the number of potential states that can be encoded into DNA memory. 56 Furthermore, distances between recombinase-recognition sites usually need to be several hundred 57 base pairs to achieve efficient recombination, thus increasing circuit size (Coppoolse et al., 2005; 58 Stark, 2017). Furthermore, recombinase sites must be pre-engineered into desired target sites, 59 which is time-and labor-intensive, especially if they are to be used in the genomic context. 60 To address these limitations, we previously developed the SCRIBE DNA writing and 61 molecular recording system, which uses in vivo single-stranded DNA expression to generate 62 precise mutations that accumulate into target genomic loci as a function of the magnitude and 63 duration of exposure to an input (Farzadfard and Lu, 2014). However, this approach has been 64 limited to bacteria thus far due to the requirement for specific recombination mechanisms. 65Alternative molecular recording strategies based on Cas9 ...

Tough Hydrogel-Based Biocontainment of Engineered Organisms for Continuous, Self-Powered Sensing and Computation

“…An higher efficiency version of SCRIBE (Synthetic Cellular Recorders Integrating Biological Events) was used in this study 45,54 . The SCRIBE strain was encapsulated in tough hydrogel beads and incubated in LB media with carbenicillin (100 µg/mL), chloramphenicol (25 µg/mL), aTc (100 ng/ml), and IPTG (1 mM) at 37°C.…”

Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

“…Inspired by synthetic biology in prokaryotes (Farzadfard et al, 2019;Tang and Liu, 2018), we reasoned that an ideal mammalian system would have capabilities of both seamlessly interfacing with the mammalian genome and progressing through a series of instructions independently of the cell's state.…”

“…Some methods for prolonging the duration of spCas9 activity have been developed to expand the length of recording, either by reducing its activity (Chan et al, 2019;McKenna et al, 2016) or through self-targeting sgRNA, which encode iterative editing of their own sequence (Kalhor et al, 2017;Perli et al, 2016). A third approach is exemplified by the DOMINO system (Farzadfard et al, 2019), which demonstrates the concept of iterative genome edits to the spacer-target sequence pairs using base editors. By progressing through a series of states in a preprogrammed, stepwise manner, DOMINO added a critical ability to combine multiplexed activities as logical operations within a larger architecture (Farzadfard et al, 2019).…”

“…A third approach is exemplified by the DOMINO system (Farzadfard et al, 2019), which demonstrates the concept of iterative genome edits to the spacer-target sequence pairs using base editors. By progressing through a series of states in a preprogrammed, stepwise manner, DOMINO added a critical ability to combine multiplexed activities as logical operations within a larger architecture (Farzadfard et al, 2019).…”

Efficient Retroelement-Mediated DNA Writing in Bacteria

“…An ideal precise DNA 37 writer (a genetically-encoded device for the targeted editing of DNA in living cells) would enable 38 one to introduce any desired mutation to any desired genomic target with high efficiency and 39 without the requirement for specific cis-encoded elements or the generation of double-strand DNA 40 breaks. Despite many advances in recent years in DNA writing technologies, existing platforms in 41 bacteria (Costantino and Court, 2003;Datsenko and Wanner, 2000;Farzadfard et al, 2019; Pines 42 et al, 2015;Swingle et al, 2010;Wang et al, 2009;Yu et al, 2000) are not ideal for certain 43 applications (Table S1). For example, recombineering-based approaches enable targeted, small 44 modification of bacterial genomes but 1) they are restricted to specific conditions in which efficient 45 transformation is possible, 2) are often limited by suboptimal editing rates, and 3) are not 46 applicable to complex environments, such as bacterial communities (Costantino and Court, 2003; 47 Wang et al, 2009;Yu et al, 2000).…”