Applications of CRISPR-Cas for synthetic biology and genetic recording

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Applications of CRISPR-Cas for synthetic biology and genetic recording Florian Schmidt1 and Randall J. Platt1,2 Abstract

The epic arms race between microbes and their predators was the driving force behind the evolution and diversification of the truly remarkable microbial adaptive immune system CRISPRCas. CRISPR-Cas systems mediate defense through three stages: recording of nucleic acid species, multiplexed RNA expression and processing, and eventually RNA-guided cleavage of hostile genetic elements. The entire process is orchestrated by a plethora of effector proteins endowed with specialized functions for manipulating genetic material. Investigating this treasure trove substantially fostered the development of the RNA-guided DNA endonuclease Cas9 into a versatile molecular tool for synthetic biology and biomedicine. Here, we review the developments of Cas9 and other CRISPR-Cas components for applications in synthetic biology as well as highlight emerging CRISPR-Cas-based genetic recorders and memory devices. Addresses 1 Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland 2 Department of Chemistry, University of Basel, Petersplatz 1, 4003 Basel, Switzerland Corresponding author: Platt, Randall J ([email protected])

Current Opinion in Systems Biology 2017, 5:9–15 Edited by Danielle Tullman-Ercek and Martin Fussenegger This review comes from a themed issue on Synthetic biology (2017) For a complete overview see the Issue and the Editorial Available online 31 May 2017 http://dx.doi.org/10.1016/j.coisb.2017.05.008 2452-3100/© 2017 Elsevier Ltd. All rights reserved.

Keywords CRISPR-Cas, Cas9, Synthetic biology, Genome editing, Genetic recording.

Introduction Genome editing technologies enabling the manipulation of genetic sequences are fueling major advancements in both basic and applied research. With an expanding molecular toolkit, our capacity to manipulate DNA, elucidate causal relationships between genotypes and phenotypes, and engineer biological systems is ever expanding, holding immense promise for the future of biomedical research. The DNA revolution began with recombinant DNA and restriction enzyme technology, enabling the manipulation of DNA in a test tube. Next came a suite of discoveries enabling the manipulation of www.sciencedirect.com

genomic DNA in living cells, which includes integrases, recombinases, transposases, and site-specific DNA endonucleases (e.g. meganucleases, zinc finger nucleases, and transcriptional activator-like effector (TALE) nucleases). Now, with the microbial defense system CRISPR-Cas, we are undergoing yet another revolution in genome editing and biomedical science. Since this transformative discovery, sculpted by dozens of scientists over three decades, we now have a vast CRISPRCas-based genome editing toolkit [1e5]. With this expanded toolkit, new possibilities emerge, including DNA mutagenesis, transcriptional activation or repression, epigenetic engineering, and molecular barcoding. Here, we provide a brief introduction of the CRISPRCas system and review emerging genome editing applications in synthetic biology. In particular, we highlight how genome editing technologies can be utilized to write information into the genome, and discuss current approaches that repurpose CRISPR-Cas for lineage tracing and genetic recording of biological activity.

The CRISPR-Cas defense system The microbial adaptive immune system CRISPR-Cas evolved as a defense system against exogenous genetic material (extensively reviewed elsewhere [1e5]). In brief, CRISPR-Cas orchestrates defense in three steps, adaptation, biogenesis, and interference. During the adaptation step, short stretches of DNA or RNA derived from invading genetic elements (i.e. protospacers) are recorded into a CRISPR array with the help of associated Cas proteins. Acquired foreign sequences within the CRISPR array (i.e. spacers) are flanked by short palindromic direct repeat sequences (i.e. DRs). During the biogenesis step, the entire CRISPR array is transcribed as a single RNA molecule and subsequently processed to yield mature guide RNAs. During the interference step, the guide RNAs direct CRISPR-associated nucleases to hostile nucleic acids and mediate their destruction. The protospacer adjacent motif (PAM), a sequence present in the invading element but not the CRISPR array or guide RNA, facilitates self versus non-self discrimination. The most widely investigated protein of the CRISPR-Cas system is the RNA-guided DNA endonuclease Cas9 from Streptococcus pyogenes (SpCas9, referred to as Cas9 herein). Cas9 recognizes it’s target site via Watson-Crick basepairing between guide RNA and target DNA. Intriguingly, Cas9 is easily targeted to seemingly any DNA sequence through modification of the 20 nucleotide targeting sequence of the guide RNA. Upon target Current Opinion in Systems Biology 2017, 5:9–15

10 Synthetic biology (2017)

recognition, Cas9 induces a DNA double strand break (DSB), which is repaired by either of two alternate DNA repair pathways. The first, termed non-homologous end joining (NHEJ) is an error-prone process leading to insertions and deletions (indels) at the target site, thereby frequently disrupting the coding sequence if targeted to an exon. The second, named homology-directed repair (HDR), is a high fidelity process that utilizes a homologous recombination template matching the target site to repair the break. Thus, Cas9 allows both gene disruption and precision editing, and can be applied to the engineering of mammalian genomes [6,7]. The development and applications of Cas9-based technologies for mammalian genome editing are diverse, immense, and extensively reviewed elsewhere [1e5]. Many of these technologies have been widely adopted and adapted, resulting in a plethora of new tools for synthetic biology. These include Cas9 orthologs (e.g. StCas9 [8], SaCas9 [9], NmCas9 [10], and FnCas9 [11] as well as other Class 2 CRISPR-Cas nucleases (e.g. Cpf1/Cas12a) [12], which enable orthogonal genome editing applications. Furthermore, fusing catalytically inactive Cas9 (dCas9) to effector proteins enables transcriptional activation (CRISPRa) [13e19], transcriptional repression (CRISPRi) [19e22], epigenetic modifications [14,19,23,24] and single base editing via a cytidine deaminase [25]. Likewise, inducible systems based on a split Cas9 architecture or nuclear localization have been achieved [26e31]. Many of these tools based on the CRISPR-Cas system have replaced their predecessors in synthetic circuits due to increased orthogonality, modularity, and multiplexability.

CRISPR-Cas-based memory devices for lineage tracing and recording biological activity Cellular behaviors are complex, dynamic, and often require the integration and orchestration of multiple stimuli and responses, respectively. Currently, we rely on time point and inference experiments that often miss the dynamic and complex single cell characteristics underlying biological phenomena. Genetic recording devices hold great potential for elucidating these diverse cellular behaviors. Early work on genetic recording relied on synthetic circuits comprised of toggle switches or multiple quasistable states based on protein expression [32e34]. Later, DNA recombinases were leveraged to record transient cellular events into genomic DNA [35,36]. These early technologies lacked scalability, orthogonality, and the capacity for capturing dynamic information, and therefore, future developments combined recombinases with inducible retron expression to encode dynamic analog memory into populations of bacteria [37]. More recently, CRISPR-Cas-based Current Opinion in Systems Biology 2017, 5:9–15

systems utilizing either Cas9 or CRISPR-Cas adaptation proteins were applied as genetic recorders (Table 1), facilitating lineage tracing (Box 1), molecular barcoding, and intracellular recording of biological activity. Scartrace

To induce sequence diversification for tracing of cellular lineages in zebrafish, Junker and colleagues injected GFP transgenic zebrafish embryos at the single-cell stage with Cas9 and GFP-targeted guide RNA ribonucleoprotein complexes (Figure 1A) [39]. They discovered, that Cas9-mediated DSBs resulted in several hundred unique indels at the target site within GFP, which allowed them to trace cellular lineages during zebrafish development and caudal fin regeneration. While this method, termed Scartrace, is easily adoptable and does not require elaborate expression constructs, the information storage capacity is limited by the diversity of indel mutations at a single target site. GESTALT

An alternative technique, termed genome editing of synthetic target arrays for lineage tracing (GESTALT), expanded the information storage capacity of CRISPRCas9-based memory devices by concatemerizing multiple guide RNA target sites (Figure 1B) [40]. This allows multiple genome editing events in the same synthetic array within the same cell. Using GESTALT, McKenna and colleagues reconstructed the lineage of 200,000 cells in the adult zebrafish, and revealed that much of the organism was formed from just a few embryonic cells. One drawback of this approach however, is the fact that simultaneous editing at two target sites of the 10 barcode concatemer can result in depletion of the intervening sequence and thus in a loss of previously stored information. mSCRIBE and homing CRISPR – evolvable and selftargeting guide RNAs

Expanding the storage capacity of CRISPR-Cas9-based memory devices even further, Perli et al. and Kalhor et al. developed self-targeting guide RNAs [41] and homing guide RNAs (referred to here as Homing CRISPR) [42], respectively (Figure 1C). With these approaches, the expression cassette encoding the guide RNA also contains the PAM sequence, and is therefore self-targeted. Upon cleavage of the guide RNA expression cassette, indels are created within the targeting sequence, and yet the guide RNA remains transcribed. Therefore, the locus undergoes multiple consecutive rounds of self-targeted cleavage and mutagenesis. This process is interrupted only when the PAM sequence is deleted, the guide RNA scaffold is destroyed, or when the guide RNA targeting sequence is shortened below 16 base pairs. Extension of the targeting sequence beyond the 20 base pairs of the canonical guide RNA resulted in genetic recordings of longer durations and sequences of greater diversity. www.sciencedirect.com

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Table 1 Comparison of CRISPR-Cas-based genetic recorders. Technology

Approach

Information

Storage capacity

Applications

Features/constraints

Citation

Scartrace

Cas9 and guide RNA targeted to a genomic locus

DNA mutations

Low

Easy to implement, simple sequencing readout, single target site severely limits storage capacity

[39] Junker et al., bioRxiv 2016

GESTALT

Cas9 and guide RNA targeted to a nonexpressed synthetic construct containing 10 target sites

DNA mutations

Medium

Cellular lineage tracing of zebrafish embryo development and caudal fin regeneration Cellular lineage tracing of zebrafish embryo, adult, and tissue (brain and heart) development

[40] McKenna et al., Science 2016

mSCRIBE

Cas9 and a polymerase III-driven synthetic construct containing a selftargeted guide RNA

DNA mutations

High

Moderately easy to implement, simple sequencing readout, limited storage capacity due to loss of previous recordings via simultaneous cleavage and deletion of array Moderately easy to implement, simple sequencing readout, high storage capacity due to selftargeting strategy, sequence diversity is proportional to the intensity and duration of biological stimuli

Homing CRISPR

Cas9 and a polymerase III-driven synthetic construct containing a selftargeted guide RNA Cas9 and guide RNA targeted to a polymerase II- driven synthetic construct containing 10 target sites and a molecular barcode

DNA mutations

High

[42] Kalhor et al., “Nature Methods” 2016

DNA mutations paired with molecular barcodes

Medium

Moderately easy to implement, simple sequencing readout, high storage capacity due to selftargeting strategy Moderately easy to implement, difficult optical readout, can be combined with topological and gene expression information

Random and programmable ~33 bp DNA sequences

High

Difficult to implement, simple to readout, difficult to analyze, multiplexable and rich information content

[44] Shipman et al., Science 2016

MEMOIR

CRISPR acquisition

CRISPR-Cas Type IE acquisition elements (Cas1, Cas2 and a leaderdriven CRISPR array) and synthetic protospacer inputs

In addition to enabling lineage tracing, sequence diversity (e.g. indel mutations in a population of cells) may also be used to record biological activity if the recording is proportional to the duration and intensity of the stimulus. Towards this end, Perli and colleagues engineered a self-targeting guide RNA recording device to be sensitive to the presence of small molecules activators such as doxycycline, IPTG or even endogenous regulators like NF-kB, which was achieved by expressing Cas9 under inducible promoters. This yielded mSCRIBE: A mammalian synthetic cellular recorder integrating biological events. Intriguingly, a xenograft of mammalian cells containing such a NF-kB responsive www.sciencedirect.com

Genetic recording of small molecule stimuli or NF-kB pathway activity in cultured cells; genetic recording of NF-kB pathway activity in engineered cells implanted in mice Cellular lineage tracing of cultured cells

Cellular lineage tracing of mouse embryonic stem cells undergoing clonal expansion; genetic recording of Wnt pathway activity in mouse embryonic stem cells Recording synthetic DNA sequences into bacterial populations over time

[41] Perli et al., Science, 2016

[43] Frieda et al., Nature 2017

mSCRIBE device could readily record the magnitude and duration of lipopolysaccharide-induced inflammation in mice, highlighting the potential use of CRISPRCas9-based recording devices for the interrogation of complex immunological processes. MEMOIR

With the aim of combining lineage information with topological and gene expression information, Frieda et al., developed a memory by engineered mutagenesis with optical in situ readout (MEMOIR) device (Figure 1D) [43]. With this approach, the target locus is expressed under a polymerase II promoter and contains Current Opinion in Systems Biology 2017, 5:9–15

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Box 1. Lineage tracing

Figure 1

A Interrogating the mechanisms underlying the development of single cells to complex organisms has inspired generations of scientists. Yet deciphering the full cell lineage of a complex organism has only been achieved for C. elegans – awarded with the Nobel Prize in Physiology or Medicine in 2002. While John Sulston could decipher the nematodes cellular lineage equipped with just a microscope, the development of many organisms or tissues is too complex, fast and takes places in opaque contexts. Compared to early attempts using dyes, tracer enzymes or fluorescent proteins, encoding information into a cell’s DNA maintains the memory not only over long periods of time and cell divisions but also when the cells are disrupted [38]. Sequencing-based lineage tracing requires sequential and increasing sequence diversity within defined regions of DNA throughout time. The more diverse the sequences, the more distantly related the individual components. Leveraging this concept, natural sequence diversity is often used to reconstruct phylogenetic relationships or tumor evolution using ribosomal sequence variation or stochastic genetic aberrations, respectively. Genome editing technologies can synthetically mimic this sequence diversification process via insertion of synthetic genetic elements or induction of gradual and stochastic modifications throughout time. In the main text we review recent applications of CRISPR-Cas-based memory devices for lineage tracing.

Scartrace target sequence

genomic locus

PAM

GFP Genetic record Indels

Cas9:guide RNA

B GESTALT

Indels

C mSCRIBE & Homing CRISPR pol III

guide RNA scaffold

self-targeted guide RNA

Indels

D a guide RNA-targeted array (i.e. ten-repeat array based on PP7 stem loops) and an additional molecular barcode. Because the locus is transcribed into mRNA, they could use multiplexed single-molecule RNA fluorescence hybridization (smFISH) to identify both the mutated array and the molecular barcode. Using their approach, they mapped lineage information onto microscopy images of mouse embryonic stem cells during clonal expansion. Besides applying MEMOIR to investigate the lineage information of cells, Frieda and coworkers used it as a synthetic, molecular recording device for Wnt signaling pathway activity by transcribing the guide RNA from a promoter that is activated by Wnt. CRISPR acquisition

While the aforementioned methods of genetic recordings with CRISPR-Cas rely exclusively on the generation of indels upon Cas9-induced DNA cleavage, also the CRISPR-Cas adaptation machinery has the potential to serve as a molecular barcoding and recording device. Shipman et al. demonstrated that the adaptation machinery of the E. Coli type I-E CRISPR-Cas system could be harnessed to acquire synthetic DNA-derived spacers into a plasmid-based CRISPR array in E. Coli (Figure 1E) [44]. Strikingly, they also demonstrated acquisition of multiple spacers over time, thus fulfilling an important requirement for recordings and generation of increasing diversity throughout several rounds of cell division.

Perspective The first generation of CRISPR-Cas-based memory devices reviewed here demonstrate their future promise and potential as powerful genetic recorders. Valuable Current Opinion in Systems Biology 2017, 5:9–15

MEMIOR pol II

molecular barcode

Indels

E CRISPR acquisition leader

spacer

DR

Cas1:Cas2:protospacer

CRISPR array spacers

Genetic recording and molecular barcoding techniques based in the CRISPR-Cas system. A) Scartrace [39] utilizes Cas9-induced indels within a single genomic site for molecular barcoding and lineage reconstruction. B) GESTALT [40] utilizes Cas9-induced indels within a synthetic array containing 10 guide RNA target sites for molecular barcoding and lineage reconstruction. C) mSCRIBE [41] and Homing CRISPR [42] utilizes Cas9-induced indels within an evolving, self-targeted guide RNA expression cassette for molecular barcoding, lineage reconstruction, and recording of biological activity. D) MEMOIR [43] utilizes Cas9-induced indels within a synthetic 10× repeat array containing guide RNA target sites for molecular barcoding, lineage reconstruction, and recording of biological activity. By expressing the synthetic array with a polymerase II promoter and including an additional molecular barcode, this approach can be combined with optical readout methods (e.g. single-molecule RNA fluorescence hybridization (smFISH)). E) CRISPR acquisition [44] utilizes synthetic and random ~33 base pairs genetic sequences acquired within the CRISPR array for molecular barcoding and recording of biological activity. This is achieved by overexpressing Type I-E CRISPR-associated proteins Cas1 and Cas2 in E. Coli followed by transformation of substrates for CRISPR acquisition.

improvements to the memory devices will include increasing the storage capacity through integrating multiple or orthogonal storage devices per cell, or by devising novel targeting strategies to increase the www.sciencedirect.com

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diversity of possible mutations. Other valuable improvements to the system will be those that enable the genetic recorders to achieve a sufficient diversity and temporal resolution to meet the requirements of the experiment, which could entail establishing a time axis for synthetic circuits, genetic recording of multimodal biological phenomena, mapping the lineage of all neurons in a mammalian brain, or uniquely labeling all parent and daughter cells of an organism.

achieving an unprecedented control and understanding of diverse biological systems.

Particularly exciting will be the combination of CRISPRCas-based memory devices with other single cell phenotyping strategies, namely single-cell RNA sequencing, topological mapping [45], and other massively paralleled perturbation strategies [46e48]. In these cases, the memory device will provide a time and/or lineage axis, bolstering integrative analyses. Ultimately, we envision that future sequencing-based integrative analyses will combine multiple orthogonal molecular barcodes informing diverse biological phenomena, including lineage, gene expression, perturbation, topology, and morphology at the single-cell level. Consequently, devising novel strategies to convert biological phenomena and relationships into molecular barcodes or sequence diversity will be particularly valuable.

References

The overwhelming majority of CRISPR-Cas-based applications in biomedical research rely on effectors from the interference stage of CRISPR-Cas defense (e.g. Cas9 and Cpf1/Cas12a). Only recently have we seen adaptation machinery (e.g. Cas1 and Cas2) exploited for applications in synthetic biology [44]. This currently untapped resource will likely undergo future developments for genetic recording and potentially site specific genome tagging. Moreover, combining these strategies with RNA-acquisition [49] may provide a means to record transcriptional events into the genome, and therefore enable the retrospective reconstruction of transcriptional histories at the single-cell level. Lastly, although not reviewed here, the recently discovered RNA-guided and RNA-targeted interference Cas proteins C2c2/Cas13a and Cas13b hold immense promise. These enzymes can cleave target RNA in vitro and in bacteria [50,51], and can be applied in vitro as a molecular diagnostic tool [52]. Further exploration of these proteins and the treasure trove of undescribed CRISPRCas effectors will likely yield powerful new molecular tools. Altogether, the discovery of genome editing technologies and their application in synthetic biology have fueled progress across the biomedical sciences. We are only in the initial phases of understanding the diversity of the CRISPR-Cas immune system and realizing its future potential. We anticipate extensive further developments in this area, both in terms of the biological parts as well as their combination with other rich phenotyping strategies. This will pave the way for www.sciencedirect.com

Acknowledgements We gratefully acknowledge the entire Platt laboratory for discussion and comments; and the CRISPR-Cas and genome editing communities for their transformative discoveries, characterizations, and technological developments. This work is supported by funds from ETH Zurich and the National Centres of Competence e Molecular Systems Engineering (NCCReMSE).

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