A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

Abstract

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health. and Extensible Mammalian Modular Assembly kit (EMMA) 3 based on Golden Gate assembly reactions were proposed. Both methods use sets of modular parts with given functionality, which can be assembled directionally into transcription units (TUs) by type-IIS restriction enzymes. While useful, all three methods have important shortcomings. GMAP requires sequencing in between all cloning steps, which makes the method slow and expensive. EMMA and mMoClo provide a method to hierarchically assemble DNA vectors that encode large circuits, but fall short on shareability, integration of Cas9 sgRNA cloning, a library of tested parts, and ease of use. While in yeast and plant systems, methods for the rapid, efficient and hierarchical assembly of genetic circuits from a large library of well described modular parts [8][9][10][11] exist, we are unaware that such a cloning toolkit for mammalian systems is readily available.In this work, we adapted the modular cloning strategy (MoClo) 12 strategy to build mammalian expression vectors. The Mammalian ToolKit (MTK) is a library of over 300 parts including vetted promoters, 3'UTRs, fluorescent proteins, insulator and P2A elements that can be combined to build large genetic circuits in a rapid and efficient fashion. These vectors can be delivered to a wide range of cell types, through viral, recombinase and CRISPR/Cas9 methods.As a proof of concept, we built a hAAVS1 landing p...

Choice of selectable marker affects recombinant protein expression in cells and exosomes

“…researchers have devoted significant time, effort, and resources to improving the outcomes of mammalian cell transgenesis experiments. Much of this effort has been directed at improved vector design, resulting in the identification of many cis-acting features that affect transgene expression, including transcriptional regulatory regions (6)(7)(8)(9)(10), mRNA polyadenylation (pAn) sites (11), introns (12), mRNA export and/or translation signals (e.g. the Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE) ( 13)), and cis-acting inhibitors of gene silencing (10,14).…”

“…Much of this effort has been directed at improved vector design, resulting in the identification of many cis-acting features that affect transgene expression, including transcriptional regulatory regions (6)(7)(8)(9)(10), mRNA polyadenylation (pAn) sites (11), introns (12), mRNA export and/or translation signals (e.g. the Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE) ( 13)), and cis-acting inhibitors of gene silencing (10,14). Other studies have revealed that transgene expression can be improved by eliminating or reducing bacterial or viral sequences that may induce transgene silencing (15,16), such as by use of DNA mini-circles (17) or DNA transposons (e.g.…”

Sentinel cells enable genetic detection of SARS-CoV-2 Spike protein

“…A computational campaign was conducted to identify short protein sequences that were able to bind the Spike protein 23 , and we incorporated the lead candidate from this screen (LCB1) into our initial antigen sensor panel ( Figure 1A ). Using our Mammalian Cloning Toolkit 31 , we rapidly constructed multiple SynNotch variants with these different extracellular sensors for Spike.…”

“…The output circuit, pHR_Gal4UASpyb-TATA_tBFP_pGK_mCitrine, was a generous gift from Dr. Kole Roybal. All other plasmids were constructed using the Mammalian Toolkit (MTK) 31 , a hierarchical DNA assembly method based on Golden-Gate (GG) cloning 56 . New binding domains were domesticated into the MTK by ordering primers or gBlocks from IDT that contained the desired coding sequence with all BsaI and BsmBI restriction sites replaced with synonymous mutations and overhangs for MTK Part 3as, then ligated into the MTK part entry vector using a BsmBI GG reaction.…”

TuNR: Orthogonal Control of Mean and Variability of Endogenous Genes in a Human Cell Line

“…Lentivirus particles were generated as described previously 47 . In short, LX-HEK293T cells were seeded at approximately 50% confluency in a 6-well plate, and the following day were transfected with lentiviral vector of interest alongside packaging plasmids (pCMV-dR8.91 and pCMV-VSV-G) using Lipofectamine 3000 (Thermo) according to manufacturer's instructions.…”