PERSIST: A programmable RNA regulation platform using CRISPR endoRNases

Regulation of transgene expression is becoming an integral component of gene therapies, cell therapies and biomanufacturing. However, transcription factor-based regulation upon which the majority of such applications are based suffers from complications such as epigenetic silencing, which limits the longevity and reliability of these efforts. Genetically engineered mammalian cells used for cell therapies and biomanufacturing as well as newer RNA-based gene therapies would benefit from post-transcriptional methods of gene regulation, but few such platforms exist that enable sophisticated programming of cell behavior. Here we engineer the 5’ and 3’ untranslated regions of transcripts to enable robust and composable RNA-level regulation through transcript cleavage and, in particular, create modular RNA-level OFF- and ON-switch motifs. We show that genomically introduced transgenes exhibit resistance to silencing when regulated using this platform compared to those that are transcriptionally-regulated. We adapt nine CRISPR-specific endoRNases as RNA-level “activators” and “repressors” and show that these can be easily layered and composed to reconstruct genetic programming topologies previously achieved with transcription factor-based regulation including cascades, all 16 two-input Boolean logic functions, positive feedback, a feed-forward loop and a putative bistable toggle switch. The orthogonal, modular and composable nature of this platform as well as the ease with which robust and predictable gene circuits are constructed holds promise for their application in gene and cell therapies.

[1]  M. T. Hasan,et al.  Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Aviv Regev,et al.  RNA targeting with CRISPR–Cas13 , 2017, Nature.

[3]  Edward S Boyden,et al.  Programmable RNA-binding protein composed of repeats of a single modular unit , 2016, Proceedings of the National Academy of Sciences.

[4]  R. Kotin,et al.  Characterization of a preferred site on human chromosome 19q for integration of adeno‐associated virus DNA by non‐homologous recombination. , 1992, The EMBO journal.

[5]  C. Daniel,et al.  Therapeutic opportunities for manipulating TReg cells in autoimmunity and cancer , 2012, Nature Reviews Drug Discovery.

[6]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Eric S. Lander,et al.  C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector , 2016, Science.

[8]  R. Elble,et al.  Tet-On lentiviral transductants lose inducibility when silenced for extended intervals in mammary epithelial cells , 2016, Metabolic engineering communications.

[9]  Vanja Haberle,et al.  Eukaryotic core promoters and the functional basis of transcription initiation , 2018, Nature Reviews Molecular Cell Biology.

[10]  J. Steel.  THE TRIPLE HELIX , 2003 .

[11]  Ron Weiss,et al.  An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells , 2019, Nature Communications.

[12]  K. Helin,et al.  Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. , 2014, Molecular cell.

[13]  T. Huang,et al.  Targeted methylation of CMV and E1A viral promoters. , 2010, Biochemical and biophysical research communications.

[14]  P. Mermelstein,et al.  Opposite Effects of mGluR1a and mGluR5 Activation on Nucleus Accumbens Medium Spiny Neuron Dendritic Spine Density , 2016, PloS one.

[15]  Swapnil Bhatia,et al.  A single-layer platform for Boolean logic and arithmetic through DNA excision in mammalian cells , 2017, Nature Biotechnology.

[16]  William F Marzluff,et al.  Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4 , 2015, RNA.

[17]  G. Keller,et al.  Identification and targeting of the ROSA26 locus in human embryonic stem cells , 2007, Nature Biotechnology.

[18]  L. Banaszynski,et al.  A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules , 2006, Cell.

[19]  P. Ingham,et al.  Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis , 2016, PloS one.

[20]  Sergey A. Shmakov,et al.  CRISPR EVOLUTION C 2 c 2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector , 2016 .

[21]  Ron Weiss,et al.  Mammalian synthetic circuits with RNA binding proteins delivered by RNA , 2015, Nature Biotechnology.

[22]  Tomoaki Hara,et al.  Synthetic translational regulation by an L7Ae-kink-turn RNP switch. , 2010, Nature chemical biology.

[23]  Andreja Majerle,et al.  A bistable genetic switch based on designable DNA-binding domains , 2014, Nature Communications.

[24]  Andreja Majerle,et al.  Designable DNA-binding domains enable construction of logic circuits in mammalian cells. , 2014, Nature chemical biology.

[25]  Timothy K Lu,et al.  A multi-landing pad DNA integration platform for mammalian cell engineering , 2018, Nucleic acids research.

[26]  Samira Kiani,et al.  An enhanced CRISPR repressor for targeted mammalian gene regulation , 2018, Nature Methods.

[27]  Markus Wieland,et al.  Programmable single-cell mammalian biocomputers , 2012, Nature.

[28]  S J White,et al.  Promoters and control elements: designing expression cassettes for gene therapy. , 2004, Current gene therapy.

[29]  Minsoo Kim,et al.  A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies , 2011, Biotechnology and bioengineering.

[30]  G. Church,et al.  Cas9 gRNA engineering for genome editing, activation and repression , 2015, Nature Methods.

[31]  Itaru Imayoshi,et al.  Light Control of the Tet Gene Expression System in Mammalian Cells. , 2018, Cell reports.

[32]  L. Nissim,et al.  Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. , 2014, Molecular cell.

[33]  O. Abudayyeh,et al.  RNA editing with CRISPR-Cas13 , 2018 .

[34]  Max J. Kellner,et al.  RNA editing with CRISPR-Cas13 , 2017, Science.

[35]  Michael B Elowitz,et al.  Programmable protein circuits in living cells , 2018, Science.

[36]  R. Weiss,et al.  Multi-input Rnai-based Logic Circuit for Identification of Specific , 2022 .

[37]  A. Schambach,et al.  Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy. , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

[38]  Corey W. Liu,et al.  Small molecule displacement of a cryptic degron causes conditional protein degradation , 2011, Nature chemical biology.

[39]  Ming Shi,et al.  A microRNA-inducible CRISPR–Cas9 platform serves as a microRNA sensor and cell-type-specific genome regulation tool , 2019, Nature Cell Biology.

[40]  Dipali G. Sashital,et al.  An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3 , 2011, Nature Structural &Molecular Biology.

[41]  Jennifer A. Doudna,et al.  Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases , 2013, Nucleic acids research.

[42]  Xiaoyin Wang,et al.  The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells , 2017, Journal of cellular and molecular medicine.

[43]  Paul Shinn,et al.  HIV-1 Integration in the Human Genome Favors Active Genes and Local Hotspots , 2002, Cell.

[44]  S. Konermann,et al.  Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors , 2018, Cell.

[45]  Phillip A Sharp,et al.  A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails. , 2012, Genes & development.

[46]  Kira S. Makarova,et al.  Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28 , 2016, bioRxiv.

[47]  Bruce T. Lahn,et al.  Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter , 2010, PloS one.

[48]  D. Zhao,et al.  A facile soft-template synthesis of mesoporous polymeric and carbonaceous nanospheres , 2013, Nature Communications.

[49]  E. Li,et al.  DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease , 2017, Proceedings of the National Academy of Sciences.

[50]  Michel Sadelain,et al.  Safe harbours for the integration of new DNA in the human genome , 2011, Nature Reviews Cancer.

[51]  J. Steitz,et al.  Hoogsteen-position pyrimidines promote the stability and function of the MALAT1 RNA triple helix , 2016, RNA.

[52]  Ron Weiss,et al.  Engineering protein-protein devices for multilayered regulation of mRNA translation using orthogonal proteases in mammalian cells , 2018, Nature Communications.

[53]  R. Terns,et al.  Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. , 2008, Genes & development.

[54]  Megan L Hochstrasser,et al.  Cutting it close: CRISPR-associated endoribonuclease structure and function. , 2015, Trends in biochemical sciences.

[55]  James A. Stapleton,et al.  Feedback Control of Protein Expression in Mammalian Cells by Tunable Synthetic Translational Inhibition , 2011, ACS synthetic biology.

[56]  J. Scheller,et al.  A widespread sequence-specific mRNA decay pathway mediated by hnRNPs A1 and A2/B1 , 2016, Genes & development.

[57]  A. Das,et al.  Optimization of the Tet-On system for regulated gene expression through viral evolution , 2006, Gene Therapy.

[58]  J. Steitz,et al.  Formation of triple-helical structures by the 3′-end sequences of MALAT1 and MENβ noncoding RNAs , 2012, Proceedings of the National Academy of Sciences.

[59]  Thomas E. Gorochowski,et al.  Organization of feed-forward loop motifs reveals architectural principles in natural and engineered networks , 2017, Science Advances.

[60]  M. Bühler,et al.  The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1 , 2017, Molecular cell.

[61]  V. Kunin,et al.  Evolutionary conservation of sequence and secondary structures in CRISPR repeats , 2007, Genome Biology.

[62]  M. Gossen,et al.  Transcriptional activation by tetracyclines in mammalian cells. , 1995, Science.

[63]  Jennifer A. Doudna,et al.  Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease , 2010, Science.

[64]  Michel Sadelain,et al.  Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells , 2011, Nature Biotechnology.

[65]  Ron Weiss,et al.  A mixed antagonistic/synergistic miRNA repression model enables accurate predictions of multi-input miRNA sensor activity , 2018, Nature Communications.

[66]  William F Marzluff,et al.  Inducing circular RNA formation using the CRISPR endoribonuclease Csy4. , 2017, RNA.

[67]  Ron Weiss,et al.  A platform for rapid prototyping of synthetic gene networks in mammalian cells , 2014, Nucleic acids research.

[68]  Tina Lebar,et al.  Design of fast proteolysis-based signaling and logic circuits in mammalian cells , 2018, Nature Chemical Biology.

[69]  Albert J R Heck,et al.  Structural basis for CRISPR RNA-guided DNA recognition by Cascade , 2011, Nature Structural &Molecular Biology.

[70]  Domitilla Del Vecchio,et al.  An endoribonuclease-based incoherent feedforward loop for decoupling resource-limited genetic modules , 2019 .

[71]  Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters , 2011, Systems and Synthetic Biology.

[72]  Y. Nahmias,et al.  Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition , 2018, Stem cell reports.

[73]  M. Goll,et al.  Transgenerational analysis of transcriptional silencing in zebrafish. , 2011, Developmental biology.

[74]  Ron Weiss,et al.  A ‘poly-transfection’ method for rapid, one-pot characterization and optimization of genetic systems , 2019, Nucleic acids research.

[75]  J. Steitz,et al.  Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix , 2014, Nature Structural &Molecular Biology.

[76]  David A. Scott,et al.  Functionally diverse type V CRISPR-Cas systems , 2019, Science.

[77]  M. Yap,et al.  DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines. , 2010, Journal of biotechnology.

[78]  J. P. Ferreira,et al.  Tuning gene expression with synthetic upstream open reading frames , 2013, Proceedings of the National Academy of Sciences.