Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins. 2022

Lifang Yu, and Yadan Zhang, and Mario Andrea Marchisio
School of Pharmaceutical Science and Technology, Tianjin University.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to β-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

UI MeSH Term Description Entries
D011401 Promoter Regions, Genetic DNA sequences which are recognized (directly or indirectly) and bound by a DNA-dependent RNA polymerase during the initiation of transcription. Highly conserved sequences within the promoter include the Pribnow box in bacteria and the TATA BOX in eukaryotes. rRNA Promoter,Early Promoters, Genetic,Late Promoters, Genetic,Middle Promoters, Genetic,Promoter Regions,Promoter, Genetic,Promotor Regions,Promotor, Genetic,Pseudopromoter, Genetic,Early Promoter, Genetic,Genetic Late Promoter,Genetic Middle Promoters,Genetic Promoter,Genetic Promoter Region,Genetic Promoter Regions,Genetic Promoters,Genetic Promotor,Genetic Promotors,Genetic Pseudopromoter,Genetic Pseudopromoters,Late Promoter, Genetic,Middle Promoter, Genetic,Promoter Region,Promoter Region, Genetic,Promoter, Genetic Early,Promoter, rRNA,Promoters, Genetic,Promoters, Genetic Middle,Promoters, rRNA,Promotor Region,Promotors, Genetic,Pseudopromoters, Genetic,Region, Genetic Promoter,Region, Promoter,Region, Promotor,Regions, Genetic Promoter,Regions, Promoter,Regions, Promotor,rRNA Promoters
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000094704 RNA, Guide, CRISPR-Cas Systems A component of CRISPR-Cas SYSTEMS. Cas endodeoxyribonucleases assemble with a guide RNA complex, a hybrid of CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) molecules, to form an active complex that cleaves DNA. crRNA and tracrRNA can be synthetically fused into a single RNA molecule, namely single guide RNA. Synthetic sgRNA is used with CRISPR-Cas SYSTEMS for targeted GENE EDITING. CRISPR Guide RNA,CRISPR-Cas Systems sgRNA (Single Guide RNA),Guide RNA (CRISPR-Cas Systems),Guide RNA, CRISPR-Cas Systems,RNA, CRISPR Guide,RNA, Guide (CRISPR-Cas Systems),RNA, Single Guide,RNA, Single-Guide,Single Guide RNA,Single-Guide RNA,Transactivating crRNA,crRNA,crRNA, Transactivating,sgRNA (CRISPR-Cas Systems),sgRNA (Single-Guide RNA),tracrRNA,Guide RNA, CRISPR,Guide RNA, CRISPR Cas Systems,Guide RNA, Single
D012441 Saccharomyces cerevisiae A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement. Baker's Yeast,Brewer's Yeast,Candida robusta,S. cerevisiae,Saccharomyces capensis,Saccharomyces italicus,Saccharomyces oviformis,Saccharomyces uvarum var. melibiosus,Yeast, Baker's,Yeast, Brewer's,Baker Yeast,S cerevisiae,Baker's Yeasts,Yeast, Baker
D014157 Transcription Factors Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process. Transcription Factor,Factor, Transcription,Factors, Transcription
D053263 Gene Regulatory Networks Interacting DNA-encoded regulatory subsystems in the GENOME that coordinate input from activator and repressor TRANSCRIPTION FACTORS during development, cell differentiation, or in response to environmental cues. The networks function to ultimately specify expression of particular sets of GENES for specific conditions, times, or locations. Gene Circuits,Gene Modules,Gene Networks,Transcriptional Networks,Gene Module,Circuit, Gene,Circuits, Gene,Gene Circuit,Gene Network,Gene Regulatory Network,Module, Gene,Modules, Gene,Network, Gene,Network, Gene Regulatory,Network, Transcriptional,Networks, Gene,Networks, Gene Regulatory,Networks, Transcriptional,Regulatory Network, Gene,Regulatory Networks, Gene,Transcriptional Network
D058727 RNA, Small Untranslated Short RNA, about 200 base pairs in length or shorter, that does not code for protein. Short Noncoding RNA,Small Non-Coding RNA,Small Non-Messenger RNA,Small Non-Protein-Coding RNA,Small Noncoding RNA,Small Untranslated RNA,sncRNA,sncRNAs,Non-Coding RNA, Small,Non-Messenger RNA, Small,Non-Protein-Coding RNA, Small,Noncoding RNA, Short,Noncoding RNA, Small,RNA, Short Noncoding,RNA, Small Non-Coding,RNA, Small Non-Messenger,RNA, Small Non-Protein-Coding,RNA, Small Noncoding,Small Non Coding RNA,Small Non Messenger RNA,Small Non Protein Coding RNA,Untranslated RNA, Small
D064113 CRISPR-Cas Systems Adaptive antiviral defense mechanisms, in archaea and bacteria, based on DNA repeat arrays called CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR elements) that function in conjunction with CRISPR-ASSOCIATED PROTEINS (Cas proteins). Several types have been distinguished, including Type I, Type II, and Type III, based on signature motifs of CRISPR-ASSOCIATED PROTEINS. CRISPR Cas Systems,CRISPR-Cas System,System, CRISPR-Cas,Systems, CRISPR-Cas

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