CN116064597B

CN116064597B - Directed evolution and darwinian adaptation in mammalian cells by autonomous replication of RNA

Info

Publication number: CN116064597B
Application number: CN202310078321.2A
Authority: CN
Inventors: 马良; 林一瀚
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2023-01-17
Filing date: 2023-01-17
Publication date: 2024-04-26
Anticipated expiration: 2043-01-17
Also published as: CN116064597A

Abstract

The present invention relates to an orthogonal alphavirus RNA replication system REPLACE system to evolve RNA-based elements, enabling sustained in vivo evolution of RNA replicase-assisted mammalian cells. The method of the present invention is capable of continuous diversification and selection of over 10 billion autonomously replicating RNA copies, RNA replication by replicase restriction patterns and powerful inducible RNA mutations. The present invention evolves new functions of fluorescent proteins, transcription factors, and mini-Cas proteins (dCasMINI) or improves upon existing functions. The present invention shows that cells equipped with replacement can adapt to challenges outside or inside the cell by continually evolving autonomously replicating RNAs that carry the key genes associated with cancer (i.e., MEK1 and KRAS). The novel RNA-based evolution platform of the invention provides a novel high-performance tool box for mammalian synthetic biology and facilitates the adaptive engineering of mammalian cells and tissues.

Description

Directed evolution and darwinian adaptation in mammalian cells by autonomous replication of RNA

Technical Field

The present invention relates to the field of bioengineering, and in particular to directed evolution and darwinian adaptation in mammalian cells by autonomous replication of RNA.

Background

Although mammalian synthetic biology brings about the hope ^{7–9,21–24} for solving human health problems, it still needs to overcome two major obstacles. First, optimizing the synthesis device for a given function is laborious ^7–9 due to the complexity of the correspondence of gene sequences to functions in general. Second, the pre-set synthesis device is not only difficult to infinitely adapt to changing environments, but also may exhibit mutations ^6,8,10,25,26 that lead to loss of function. While directed evolution of synthetic devices within mammalian cells can help address these challenges: on the one hand, the construction of the synthesis apparatus can be accelerated by directed evolution, i.e. even if the genes or gene lines are diversified (e.g. by induced mutation) and then screened ^27–30; on the other hand, the ability of the synthesizer to evolve, particularly continuously within living cells, will allow the cells to evolve and adapt to environmental changes, or reverse the deleterious variations ^10,25,26 that occur in the synthesizer.

Just as with yeast-based directed evolution method OrthoRep, ³¹, an ideal mammalian cell-based directed evolution method should also isolate the synthetic device from the host genome to ensure continuous, orthogonal and long-term evolution. It also needs to overcome the difficulty of inefficient transfection and slow ¹ proliferation of mammalian cells, which makes it difficult to form large-scale libraries of mutants within mammalian cells. Further, a relatively broad mutation spectrum is necessary to form a complete sequence-to-function mapping map for the synthesis apparatus, while also improving the performance ^1,3 of the method. Furthermore, achieving adjustable control of mutation rate ³ is very beneficial, as it will adapt to various application scenarios by adjusting the mutagenesis time and mutagenesis duration. However, the existing method ^1,2,11–19 does not meet all of the above requirements.

Disclosure of Invention

Unlike the usual methods of evolving DNA-based devices, the inventors have chosen to evolve RNA in mammalian cells, taking into account a number of factors. First, the prospect of RNA-based therapies is very optimistic: RNA can evolve orthogonally due to its ease of entry into mammalian cells and tissues, and very low interference ^32,33 with the host genome. Second, mammalian cells can stably carry many copies ³⁴ of foreign RNAs, providing a means of constructing and screening large libraries of variants. Third, RNA can replicate ^35,36 in mammalian cells by replicase enzymes of RNA viruses, and the accuracy of its replication can be regulated. By taking the characteristics of the RNA into consideration, the inventor herein developed a REPLACE (RNA REPLICASE-assisted continuous in vivo evolution) technology, a directed evolution technology based on autonomously replicating RNA using mammalian cells as the chassis cells. It ensures orthogonality, constructs a relatively large-scale library of mutants, provides a relatively balanced and broad mutation spectrum, allows control of mutagenesis rates, and operates in continuous or discontinuous modes. The inventors herein demonstrate a series of application scenarios for REPLACE. It will become a versatile, powerful synthetic biology tool for direct adaptation to mammalian biomacromolecule engineering and to provide synthetic evolution capabilities in mammalian cells and tissues.

In one aspect, the invention provides an RNA replicase-assisted continuous in vivo evolution (REPLACE) system comprising autonomously replicating RNA and a host cell, optionally further comprising a mutagen that induces mutation.

In one embodiment, the autonomously replicating RNA lacks an RNA replicase and the host cell constitutively expresses the deleted RNA replicase.

In another embodiment, the autonomously replicating RNA is derived from a positive-stranded RNA virus, preferably from an alphavirus, more preferably from a Sindbis virus, more preferably from repRNA-v1, more preferably from repRNA-v2.

In yet another embodiment, the autonomously replicating RNA is selected from any one of repRNA-v3, repRNA-v4 or is derived from any one of repRNA-v3, repRNA-v4, preferably the autonomously replicating RNA is repRNA-v4 or is derived from repRNA-v4, more preferably the autonomously replicating RNA is repRNA-v4.

In another embodiment, the host cell is a eukaryotic host cell, preferably the host cell is an animal host cell, more preferably the host cell is a mammalian host cell, more preferably the host cell is selected from or derived from a hamster fibroblast cell line, more preferably the host cell is selected from or derived from BHK-21 (ATCC), more preferably the host cell is a replicase-limited eukaryotic host cell, preferably the host cell is a replicase-limited animal host cell, more preferably the host cell is a replicase-limited mammalian host cell, more preferably the host cell is selected from or derived from a replicase-limited hamster fibroblast cell line, more preferably the host cell is selected from or derived from a replicase-limited BHK-21 (ATCC).

In yet another embodiment, the mutagen is selected from any of a small molecule mutagen, a nucleoside analog, optionally any of the mutagens is selected from FAVIPIRAVIR, MOLNUPIRAVIR.

In another aspect, the invention provides a method of RNA replicase-assisted continuous in vivo evolution (REPLACE), comprising: providing autonomously replicating RNA; providing a host cell; continuously in vivo evolving autonomously replicating RNA; optionally also provided are mutagens that induce mutations.

In another embodiment, the host cell is a eukaryotic host cell, preferably the host cell is an animal host cell, more preferably the host cell is a mammalian host cell, more preferably the host cell is selected from hamster fibroblast cell line or derived from hamster fibroblast cell line, more preferably the host cell is selected from BHK-21 (ATCC) or derived from BHK-21 (ATCC), more preferably the host cell is a replicase-limited eukaryotic host cell, preferably the host cell is a replicase-limited animal host cell, more preferably the host cell is a replicase-limited mammalian host cell, more preferably the host cell is selected from replicase-limited hamster fibroblast cell line or derived from replicase-limited hamster fibroblast cell line, more preferably the host cell is selected from replicase-limited BHK-21 (ATCC) or derived from any of replicase-limited BHK-21 (ATCC).

In a further aspect, the invention provides the use of a REPLACE system of the invention in the manufacture of a biomacromolecule, vaccine, medicament, in the generation of a mutant pool or in the realization of darwinian adaptation.

In another aspect, the invention provides a vector for expressing the autonomously replicating RNA of the invention.

In yet another aspect, the invention provides a host cell comprising an expression vector for expressing an autonomously replicating RNA of the invention.

In another aspect, the invention provides a vaccine composition comprising RNA produced by or producible by the REPLACE system of the invention.

In yet another aspect, the invention provides a pharmaceutical composition comprising RNA produced by or producible by the REPLACE system of the invention.

In another aspect, the invention provides a delivery vehicle comprising RNA produced by or producible by the REPLACE system of the invention.

The invention has the beneficial effects of ensuring orthogonality, constructing a relatively large-scale mutant library, providing a relatively balanced and wide mutation spectrum, allowing control of mutagenesis rate, and operating in a continuous or discontinuous mode.

Drawings

FIG. 1 is a schematic representation of self-replicating RNA in mammalian cells. These two self-replicating RNAs are from literature ^42,43, in which candidate RNA variants exhibit reduced cytopathological characteristics (see methods of the application for details).

FIG. 2 is a quantification of EGFP signal expressed 24 hours after electroporation by flow analysis, and a quantification of cell numbers of designated autonomously replicating RNAs (under selection of 10. Mu.g/ml puromycin) 10 days after electroporation. P values were from a two-tailed t-test, error bars represent standard deviation (n=3 biological replicates).

FIG. 3 is a library screening experiment of self-replicating RNA in mammalian cells. RNA libraries (repRNA-v 2 based) capped and tailed with polyadenylation were electroporated into wild-type BHK-21 cells. The penultimate amino acids of nsP1 and nsP2 are encoded by NNK codons. Representative images of EGFP (left) and bright field (right) show cells 7 days after electroporation.

FIG. 4 is a graph of the quantification of the relative proportion of amino acids corresponding to each NNK codon of the RNA library before and after the screening in FIG. 3. Error bars represent standard deviation (n=3 biological replicates).

FIG. 5 is a construction and characterization of autonomously replicating RNA with enhanced host compatibility in mammalian cells. a, designing a library screening experiment of virus autonomous replication RNA in mammalian cells. RNA containing nsP1-3 and a coding box driven by a Subgenomic (SG) promoter was replicated in cells expressing nsP 4. The penultimate two amino acids of nsP1 and nsP2 are encoded in the NNK codon. b, representative EGFP (top) and bright field images (bottom) show cells 7 days after library electroporation.

FIG. 6 is a graph of the quantification of cell numbers 7 days after screening of two RNA libraries (pool 1 refers to FIGS. 3-4, and pool 2 refers to FIGS. 5 and 7). P values were from a two-tailed t-test, error bars represent standard deviation (n=3 biological replicates).

FIG. 7 is a graph quantifying the relative proportion of individual amino acids for each NNK codon in the mutant library before and after screening. Error bars represent standard deviation (n=4 biological replicates).

Fig. 8 is a representative picture of three versions of autonomously replicating RNA and their corresponding EGFP (top) and bright field (bottom) taken 1 day and 7 days after electroporation into host cells, respectively.

FIG. 9 is the number of cells quantified for different RNA versions 7 days after electroporation. P values were from a two-tailed t-test, error bars represent standard deviation (n=3 biological replicates).

FIG. 10 is a test design to quantify and compare short term stability of RNA by measuring EGFP expressed by the RNA at an early point in time of entry into the cell.

FIG. 11 is a flow cytometer quantitative analysis of EGFP and iRFP fluorescence of cells electroporated with different versions of RNA at designated time points. Note that only repRNA-v4 of the host cells expressed the iRFP signal (i.e., expressed as replicase levels).

FIG. 12 is a quantification of EGFP signal (including the proportion of EGFP-positive cells and the average EGFP intensity of EGFP-positive cells) for two versions of autonomously replicating RNA over 3 days (repRNA-v 2; repRNA-v 3). n=2 biological replicates.

FIG. 13 is a quantification of EGFP signal (including the proportion of EGFP-positive cells and the average EGFP intensity of EGFP-positive cells) by one version of autonomously replicating RNA over 3 days (repRNA-v 4). Cells with different levels of iRFP signal were separately sorted and quantified to compare their behavior at different replicase levels. n=2 biological replicates.

FIG. 14 is a pathway enrichment analysis of genes upregulated in cells carrying repRNA-v3 compared to cells carrying repRNA-v4 (see methods for details).

FIG. 15 is a graph of short term RNA stability quantified by relative changes in EGFP-positive portions 36 hours and 72 hours post electroporation. repRNA-v4 quantification was performed in two hosts, where cells from either host were clustered by replicase level (iRFP), and the proportion of EGFP-positive cells was calculated for each population separately.

FIG. 16 is a schematic of a low replicase host carrying repRNA-v 4. The low replicase host greatly reduced its expression by inserting a stop codon in front of the nsP4 gene compared to the high replicase host (FIGS. 5a, 10 and detailed methods).

FIG. 17 is a quantification of EGFP signal (including the proportion of EGFP-positive cells and the average EGFP intensity of EGFP-positive cells) in 3 days for low replicase host cells carrying repRNA-v4 (as shown in FIG. 10). Cells with different levels of iRFP signal were separately clustered and quantified to compare behavior at different replicase levels, and the data was also used in figure 15.

FIG. 18 is a scatter plot showing steady state EGFP levels and corresponding replicase levels for each cell population of FIG. 15. The red line represents a linear fit.

FIG. 19 is the result of quantifying EGFP and iRFP signals of repRNA-v4 high replicase host cells using FACS (3 weeks post electroporation) and the mean iRFP signal level of the data therein (i.e., representative of replicase level), error bars represent standard deviations (n=3 biological replicates).

Fig. 20 is a quantification of a low replicase host similar to fig. 19 and the mean iRFP signal level of the data therein, error bars represent standard deviation (n=3 biological replicates).

Fig. 21 is a graph of long-term RNA stability (n=3 biological replicates) quantified by changes in EGFP positive fractions during one month of culture using a population of cells that has been stable after a period of culture (2 weeks after electroporation).

FIG. 22 is a one month time scale quantification of EGFP and iRFP signals of repRNA-v4 carrying high replicase host cells using streaming. The data is quantized and presented in fig. 21.

FIG. 23 is a representative image showing the superposition of dsRNA signals (red, immunostained signals) and nuclear signals (blue, hoechst) of repRNA-v4 host cells.

FIG. 24 is a box plot showing quantification of dsRNA signals from immunostaining experiments in FIG. 23 (detailed in methods).

FIG. 25 is the result of the quantification of RPKM for Gapdh and nsP1-nsP3 by RNA sequencing of repRNA-v4 host cells (n=2 biological replicates).

FIG. 26 is a graph of the controlled RNA mutagenesis achieved by modulating the fidelity of RNA replicase using nucleoside analogs. a, experimental design of mutation analysis of replication RNA in the absence of disturbance (i.e. in basal conditions). b, total mutation frequency of the target region (nsP 1-3) on plasmid DNA or autonomously replicating RNA at the indicated time point after electroporation. Error bars represent standard deviation (n=3 biological replicates).

FIG. 27 is a scatter plot showing total mutation frequency versus days of replication in host cells. The data is from b of fig. 26. Asterisks indicate the average of 3 experiments. The red line represents a linear fit to the data.

FIG. 28 is a mutation profile of IVT RNAs and autonomously replicating RNAs in cells 21 days after electroporation. Error bars represent standard deviation (n=3 biological replicates). p values were calculated by a two-tailed t-test (< 0.05, <0.01, and < 0.001).

FIG. 29 is a characterization of the mutant ability of nsP 4C 488G. Left panel, comparison of total mutation frequencies of target regions (nsP 1-3) 10 days after electroporation of wild-type nsP4 and nsP 4C 488G mutants. Right panel, comparison of mutation spectra. P values were calculated by a two-tailed t-test, error bars represent standard deviation (n=3 biological replicates).

FIG. 30 shows EGFP fluorescence levels after 72 hours of treatment with ribavirin and 5-AzaC at the indicated concentrations. Error bars represent standard deviation (n=3 biological replicates).

FIG. 31 is a FACS quantification of fluorescence intensity of EGFP from cells treated with nucleoside analogs molnupiravir and favipiravir at indicated concentrations for 72 hours. The upper right panel is a representative picture showing a decrease in EGFP fluorescence after molnupiravir treatment. Error bars represent standard deviation (n=3 biological replicates).

FIG. 32 is the total mutation frequency of the target region (nsP 1-3) on the replicated RNA after treatment with molnupiravir or favipiravir for 72 hours at the indicated concentrations.

FIG. 33 is EGFP signal distribution of repRNA-v4 carrying cells after 72 hours of treatment at the indicated concentrations of molnupiravir or favipiravir.

FIG. 34 is a distribution and corresponding bar graph of EGFP signals after cells carrying repRNA-v4 were treated by molnupiravir or favipiravir over the course of 72 hours. Error bars represent standard deviation (n=3 biological replicates), and total mutation frequency of the target region on replicated RNA treated with molnupiravir or favipiravir over 72 hours. Error bars represent standard deviation (n=3 or 6 biological replicates).

FIG. 35 is a distribution of iRFP signals of repRNA-v 4-carrying host cells after 72 hours of treatment at the indicated concentrations of molnupiravir or favipiravir; distribution of the iRFP signal by host cells carrying repRNA-v4 on a 72 hour time scale treated with molnupiravir or favipiravir; and quantification of constitutively expressed EGFP signals in control cell lines after 72 hours of treatment at either the indicated concentrations molnupiravir or favipiravir. Error bars represent standard deviation (n=3 biological replicates).

FIG. 36 is a mutation profile of molnupiravir or favipiravir-induced autonomously replicating RNA. n=6 (DMSO ctrl) or n=3 (other conditions) biological repeats. p values were calculated by a two-tailed t-test (< 0.05, <0.01, and < 0.001).

FIG. 37 is a design of autonomously replicating RNA carrying EGFP and corresponding host cells.

FIG. 38 is a plot of the evolution experiment timeline for blue-shifted EGFP and corresponding quantification of flow analysis at a designated time point; and mutation profile of EGFP mRNA after 2 days molnupiravir of treatment. The high baseline mutation frequency (e.g., DMSO conditions) is due to the cells being cultured for a long period of time prior to the experiment. Error bars represent standard deviation (n=3 biological replicates). P values were calculated by a two-tailed t-test (< 0.05, <0.01, and < 0.001).

FIG. 39 is a nucleotide level mutation analysis of EGFP mRNA in a sorted cell population. Mutations at both the RNA level and the protein level are marked. Details of sorting are shown in the middle of fig. 38.

Figure 40 is the percentage of the mutations in different cell populations. Error bars represent standard deviation (n=3 technical replicates).

FIG. 41 is a flow quantitative analysis of wild-type EGFP and two evolved mutants (Y66H, T203I), demonstrating one data in three biological replicates.

FIG. 42 is a transcription factor of a response ligand synthesized using REPLACE engineering. a, design of autonomously replicating RNA carrying TetR-VP48 and host cells containing nsP4 and homologous TRE3G reporter genes. Note that this autonomously replicating RNA is similar to repRNA-v4, the penultimate amino acid of both nsP1 and nsP2 being valine. b, time line of experimental design and TetR-VP48 directed evolution. See the method for details.

FIG. 43 is a nucleotide level mutation analysis of TetR mRNA in a sorted cell population (i.e., 1% of cells with highest mCherry signals).

FIG. 44 is the fluorescence intensity of the reporter gene activated by wild-type TetR-VP48 and the evolution mutant at the indicated doxycycline (Dox) concentration. Blue represents the fold change in reporter system signal with and without ligand. Error bars represent standard deviation (n=3 biological replicates).

FIG. 45 is the design of autonomously replicating RNA carrying VP64-PadR, as well as the design of a host cell containing nsP4 and a reporter gene driven by a promoter containing the PadR binding site; and a timeline of experimental design and VP64-PadR directed evolution. See the method for details.

FIG. 46 is a nucleotide level mutation analysis (no sorting) of PadR mRNA in the resulting cell population.

FIG. 47 is the fluorescence intensity of the reporter gene activated by wild-type VP64-PadR and the evolved mutant at the indicated sodium ferulate concentration. Blue represents the fold change in the reporter system fluorescence signal with and without ligand. Error bars represent standard deviation (n=3-6 biological replicates).

FIG. 48 is the fluorescence intensity of reporter genes activated by wild-type VP64-PadR and evolution mutants at the indicated sodium ferulate concentrations. The mutant selected is particularly interesting because it exhibits an opposite ligand sensitivity compared to the wild type, which is also quantified in fig. 47. Error bars represent standard deviation (n=3 biological replicates).

FIG. 49 is a time line of experimental design and directed evolution of VP64-PadR in the absence of ligand (i.e., ligand-free evolution). In this experiment, autonomously replicating RNA and host cells in fig. 45 were used. See the method for details.

FIG. 50 is a nucleotide level mutation analysis of PadR mRNA obtained by flow clustering in the ligand-free evolution experiment of FIG. 49.

FIG. 51 is the fluorescence intensity of the reporter gene activated at the sodium ferulate concentration shown for wild-type VP64-PadR and the evolved mutant (from ligand-free evolution experiments). The fold change in reporter gene expression for each variant is indicated in blue. Error bars represent standard deviation (n=3 biological replicates). P values were calculated by a two-tailed t-test (< 0.05, <0.01, and < 0.001).

FIG. 52 is a ligand dose dependence of wild-type VP64-PadR and VP64-PadR (D21G/D74V/M178T) mutants and the resulting dose response curves fitted. The resulting Hill coefficient and semi-inhibitory concentration (IC 50) of each material fit are indicated. Error bars represent standard deviation (n=3 biological replicates).

FIG. 53 is the fluorescence intensities of wild-type dCasMINI-VPR and dCasMINI (S246R) -VPR activated TRE3G reporter genes. Fold change between reported levels for mutant and wild type is indicated in blue. Error bars represent standard deviation (n=5 biological replicates).

FIG. 54 is a graph showing that the quantification of CD2 is similar to h (staining with antibodies). Ctrl indicates no sgRNA control. Error bars represent standard deviation (n=4 biological replicates). P values were calculated by a two-tailed t-test (< 0.05, <0.01, and < 0.001).

FIG. 55 is a graph showing base editing frequencies at three endogenous sites in HEK293T cells after editing based on wild-type dCasMINI or mutant dCasMINI (S246R). Error bars represent standard deviation (n=4 biological replicates). P values were calculated by a two-tailed t-test (< 0.05 and < 0.01). And CasMINI or mutant CasMINI (S246R) cleavage frequencies at four endogenous sites in HEK293T cells. Error bars represent standard deviation (n=3 biological replicates). Wherein, the sgRNA is the sgRNA of literature ²⁰.

Fig. 56 is an experimental design to test whether an RNA carrying MEK1 could adapt cells to challenges of therapeutic drugs.

Fig. 57 is a summary of the mutations identified on MEK1 mRNA in cell populations that are adapted to three different MEK1 inhibitors. There are three biological replicates for each inhibitor.

Fig. 58 is a mutation analysis of MEK1 mRNA in a cell population adapted to three different MEK1 inhibitors. a, mutation analysis of the nucleotide level of MEK1 mRNA in a cell population collected prior to replacement experiments. b, nucleotide level mutation analysis of MEK1 mRNA in cell populations that are compatible with cobicitinib challenge. c, nucleotide level mutation analysis of MEK1 mRNA in cell populations that are adapted to trimetinib challenge. d, nucleotide level mutation analysis of MEK1 mRNA in a cell population that is adapted to the semtinib challenge.

Fig. 59 is a summary of mutations identified along the domain of MEK 1. DD: a docking domain; NES: a core outputs a signal; NRR: a negative regulation region; KCD: a kinase catalytic domain; AL: an activation ring; DVD: multifunctional docking domain. The comments regarding the domains are adapted from document 60.

FIG. 60 is the fluorescence intensity of SRE reporter genes activated by wild type MEK1 and evolved mutants under the indicated conditions. Error bars represent standard deviation (n=3 biological replicates).

Figure 61 is a design of experiment to test whether KRAS-carrying RNAs can adapt cells to the challenges inherent in cells (i.e., dominant suppressed KRAS alleles).

FIG. 62 is a summary of mutations identified on KRAS (S17N) mRNA in cell populations adapted for dominant suppression. For ease of comparison, the graphs also contain corresponding mutation information for experiments using wild-type KRAS. Three biological replicates were run for each replicated RNA.

Fig. 63 is a further feature of KRAS adaptation experiments. a, nucleotide level mutation analysis of KRAS (wild-type) mRNA in cell populations collected prior to replacement experiments. b, nucleotide level mutation analysis of KRAS (wild type) mRNA in the cell population collected on day 14. c, nucleotide level mutation analysis of KRAS (S17N) mRNA in cell populations collected prior to replacement experiments. d, nucleotide level mutation analysis of KRAS (S17N) mRNA in cell populations escaping dominant suppression.

FIG. 64 is a summary of mutations identified along the domain of KRAS. P-loop: a phosphate binding ring; α3: alpha helix 3; HVR: high variation region. The comments regarding the domains are adapted from document 69.

Figure 65 is the fluorescence intensity of SRE reporter gene activated by KRAS variant (top) and the corresponding effect on cell proliferation expressed by cell number relative to baseline (bottom). Error bars represent standard deviation (n=5 or 6 biological replicates). For (d, h), p values were calculated by a two-tailed t-test (< p <0.05, < p <0.01, and, < p < 0.001).

Figure 66 is the fluorescence intensity of SRE reporter gene activated by KRAS variant (top) and the corresponding effect on cell proliferation of cell number relative to baseline (bottom). Error bars represent standard deviation (n=5-6 biological replicates). P values were calculated by a two-tailed t-test (×p < 0.001).

FIG. 67 is a design of a replication RNA carrying dCasMINI-VPR and a nsP 4-containing host cell, the reporter gene driven by the TRE3G promoter, while the sgRNA driven by the U6 promoter is directed against the TetO site on the TRE3G promoter.

FIG. 68 is a directed evolution timeline of experimental design and dCasMINI-VPR. See the method for details.

FIG. 69 is a nucleotide level mutation analysis (no sorting) of DCASMINI MRNA in a harvested cell population.

FIG. 70 is a graph showing the effect of varying concentrations of nucleoside analogs on cell proliferation, as quantified relative to the number of DMSO-treated cells. Error bars represent standard deviation (n=3 biological replicates).

Detailed Description

The present invention can be carried out by the following embodiments, but the present invention is not limited thereto.

Definition of the definition

As used herein, the term "RNA replicase" refers to RNA-dependent RNA polymerases that replicate RNA having a specific secondary structure as a template.

As used herein, the term "autonomously replicating RNA" or "self-replicating RNA" refers to RNA that is capable of self-replication by the action of an RNA replicase, using its RNA sequence as a template.

As used herein, the term "host cell" refers to a chassis cell capable of carrying autonomously replicating RNA.

As used herein, the term "replicase-limited host cells" refers to host cells that express moderate amounts of RNA replicase and maintain stable self-replicating RNA for long periods of time.

As used herein, the term "mutagen" refers to a substance that causes a change in the genetic material of a cell that causes its genetic mutation or chromosomal aberration to a level above natural.

As used herein, the term "small molecule mutagen" refers to a mutagen of the chemical small molecule type.

As used herein, the term "nucleoside analog" refers to a structurally modified nucleoside (the portion of a nucleotide that contains only five carbon sugars and bases).

As used herein, the term "positive-strand RNA virus" refers to a virus that uses positive-strand RNA as a direct mRNA, translates early proteins, and then replicates to form a complementary strand (negative strand) under the action of RNA replicase using the positive-strand RNA as a template.

As used herein, the term "alphavirus" refers to a class of viruses that are classified as Alphavirus on a viral taxonomic basis.

As used herein, the term "Sindbis virus" is a positive strand RNA virus called Sindbis virus, hereinafter referred to as the genus hendbis virus.

As used herein, the term "repRNA-v1" refers to the replicative RNA obtained after substitution of proline to leucine at position 726 of nsP2 of Sindbis virus and structural protein to EGFP-T2A-Puro.

As used herein, the term "repRNA-v2" refers to the replicative RNA obtained after substitution of glycine at 539 of nsP1 for Sindbis virus with valine, proline at 726 of nsP2 with leucine, glycine at 806 of nsP2 with valine, and structural proteins with EGFP-T2A-Puro.

As used herein, the term "repRNA-v3" refers to the replicative RNA obtained after substitution of glycine at 539 of nsP1 for Sindbis virus with alanine, proline at 726 of nsP2 with leucine, glycine at 806 of nsP2 with isoleucine, and structural proteins with EGFP-T2A-Puro.

As used herein, the term "repRNA-v4" refers to the replicative RNA obtained after substitution of glycine at 539 of nsP1 for alanine, proline at 726 for leucine, glycine at 806 for nsP2 for isoleucine, deletion of nsP4 from the replicative RNA, and subsequent expression of nsP4 in host cells driven by the EF 1. Alpha. Promoter, with structural protein replaced with EGFP-T2A-Puro, of Sindbis virus.

As used herein, the term "evolution" refers to the variation of genetic traits in a population from generation to generation at the cellular level.

As used herein, the term "directed evolution" refers to the process of artificially making a large number of mutations in a laboratory, giving a selection pressure in a specific direction according to a specific purpose, screening protein molecules having desired characteristics, and achieving a simulated evolution at the molecular level.

As used herein, the term "in vivo evolution" refers to the process of making mutations to evolution that occur entirely within a cell.

As used herein, the term "continuous in vivo evolution" refers to the process of experiments in which cells from a previous round of screening do not require extraction of genetic material for in vitro mutagenesis, and can be used directly for the next round of mutagenesis after waiting for cell expansion.

As used herein, the term "vector" refers to a piece of nucleic acid material that can transfer genetic material from one cell to another for propagation, expression, or isolation, among other requirements.

As used herein, the term "prokaryotic vector" refers to a prokaryotic expression vector that is capable of carrying an inserted exogenous nucleic acid sequence into a prokaryotic cell for expression.

As used herein, the term "eukaryotic vector" refers to eukaryotic expression vectors, and in particular, vectors that carry an inserted exogenous nucleic acid sequence into eukaryotic mammalian cells for expression.

As used herein, the term "viral vector" refers to a vector based on a virus that can carry an inserted exogenous nucleic acid sequence into mammalian cells for expression.

As used herein, the term "self-replicating vector" refers to a vector that can replicate autonomously in a mammalian host cell.

As used herein, the term "manufacture of a biological macromolecule" refers to a process of engineering a biological macromolecule such as a protein, nucleic acid, etc., using directed evolution to meet a particular need or purpose of use.

As used herein, the term "vaccine" refers to a biological product for vaccination made with various types of pathogenic microorganisms or biological macromolecules such as their partial proteins or nucleic acids.

As used herein, the term "drug" refers to a substance that is capable of preventing, treating, and diagnosing a disease.

As used herein, the term "mutant" refers to a nucleic acid molecule that has a mutation in some bases.

As used herein, the term "mutant pool" refers to a population of a large number (often hundreds of millions) of nucleic acid molecules with mutations in certain bases.

As used herein, the term "darwinian adaptation" refers to the adaptation of cells to a new environment under the selection pressure provided by the environment, using the REPLACE system to provide mutations when the cells encounter a change in external environment, similar to the natural selection process.

As used herein, the term "orthogonal" refers to the process by which the host cell genome and the replacement system introduce mutations independent of each other.

As used herein, the term "fluorescent protein" refers to a class of proteins that have a barrel structure that itself can emit fluorescence.

As used herein, the term "transcription factor" refers to a DNA binding protein that specifically interacts with cis-acting elements of a gene and has an activating or inhibiting effect on transcription of the gene.

As used herein, the term "Cas protein" refers to shorthand for CRISPR (clustered regularly interspaced short palindromic repeats) related proteins.

As used herein, the term "mini-Cas protein" refers to a Cas protein having a protein sequence shorter in length than classical Cas9, and about several hundred amino acids in length.

As used herein, the term "cancer-associated key gene" refers to MEK1 and KRAS as used herein.

As used herein, the term "evolution platform" refers to a technology that can perform directed evolution on biological macromolecules.

As used herein, the term "DNA-based evolutionary platform" refers to directed evolution techniques that introduce mutations at the DNA level and construct a library of mutants.

As used herein, the term "RNA-based evolution platform" refers to directed evolution techniques that introduce mutations at the RNA level and construct a library of mutants.

As used herein, the term "synthetic biology" refers to engineering disciplines that purposefully design, engineer, or even resynthesize organisms or cells to have specific biological functions and that may be of value in the medical, agricultural, industrial, and environmental fields.

As used herein, the term "engineering" refers to the process of altering the sequence of an existing biological macromolecule, optimizing its function to meet the specific use needs of a person.

It is to be understood that other terms appearing herein are intended to be interpreted in accordance with their ordinary meaning in the art as understood by those skilled in the art. In the event that a term defined herein conflicts with a corresponding definition in other documents in the art, the term defined herein controls.

Method of

Construction of plasmids

The plasmids used in this study were constructed by assembly using Gibson or restriction ligation. Primers for cloning or sequencing were ordered from GENEWIZ. Amplification of DNA or cDNA fragments was performed using KOD One ^TM PCR MASTER Mix (TOYOBO, # KMM-101). Plasmid in E.coli Strain5 A (TSINGKE, TSC-C01) was maintained and amplified, extracted with HiPure PLASMID DNA MINI KIT (Magen, #P 100103) and finally confirmed by Sanger sequencing (RUIBO).

The relevant plasmid information is as follows:

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Cell culture

Hamster fibroblast cell line BHK-21 (ATCC) and human cell line HEK293T (ATCC) were used in this study. BHK-21 cells were cultured in MEM/EBSS medium (HyClone, # SH 3002401) supplemented with 10% FBS (excel, # FSP 500), 100 units/mL penicillin (penicillin), 100 μg/mL streptomycin (streptomycin), and 1% MEM nonessential amino acid solution (Gibco, # 11140050). HEK293T cells were cultured in DMEM medium (Gibco, # C11995500 BT) containing 10% FBS (excel, # FSP 500), penicillin 100 units/mL and streptomycin 100 μg/mL. All cell cultures were placed in humidified incubator at 37℃with 5% CO ₂ and routinely tested for mycoplasma.

Plasmid transfection and construction of stable cell clones

Plasmid transfection was performed using Lipofectamine ^TM LTX reagent with PLUS ^TM reagent (Thermofisher # 15338100) or PEI reagent (polyethylene glycol), 1. Mu.g/. Mu.L). The PiggyBac (PB) transposon system or lentiviral packaging system is used for plasmid stable integration. To construct a replicase-limited replicated host cell line (i.e., a BHK-21 cell line stably expressing Sindbis virus RNA-dependent RNA polymerase (nsP 4) on the BHK-21 genome), 2.5. Mu.g of the pEF1α -iRFP-NLS-P2A-. DELTA.nsP 3-nsP4 plasmid (or pEF1α -iRFP-NLS-TAG-. DELTA.nsP 3-nsP4 for a cell line with reduced expression of nsP 4) was co-transfected with 0.5. Mu. gPiggyBac transposase plasmid into one well of a 6-well plate containing BHK-21 cells, according to the instructions of Lipofectamine ^TM LTX kit, with a cell confluency of 60%. The medium was changed 12 hours after transfection. After one week of culture, iRFP positive BHK-21 cells were enriched by Fluorescence Activated Cell Sorting (FACS) using a BD FACSAria ^TM III cell sorter. To report the activity of ligand-controlled synthetic transcription factors or Cas-based transcriptional regulatory proteins, the corresponding reporter plasmid was packaged into lentiviruses and the resulting viral particles were used to transfect replicase-limited BHK-21 cell lines. Specifically, for TetR-VP48, the plasmid pTRE3G-puroR-P2A-NLS-mCherry-BGHployA-pEF1α/HTLV-mTagBFP2-NLS-WPRE was used; for PadR-VP64, plasmids (O _PadR)₆ -phCMVmin-puroR-NLS-mCherry-hPEST-BGHployA-pEF1α/HTLV-mTagBFP-NLS-WPRE; and for dCasMINI-VPR, plasmid pTRE3G-puroR-P2A-NLS-mCherry-BGHployA-phU6-sgRNA_for_TetO-pEF1α/HTLV-tagBFP-NLS-WPRE. For viral packaging, HEK293T cells were cultured to 70% cell confluence in one well of the 6 well plate and transfected with 3 μg reporter plasmid, 2.66 μg pCMVDR8.91 plasmid and 0.34 μg PMD2.G plasmid as indicated by the PEI reagent manual, and after 48 hours the medium was replaced with 2mL fresh medium, the supernatant containing viral particles was centrifuged at 500G for 3 minutes and filtered through a filter of 0.45 μg filter to introduce 500 μg of supernatant into the well plate for confluence of BHK-21. Mu.4 days after transfection into the well plate, and after transfection of BHK-21. Mu.g of PMD2.G plasmid was used for positive cell separation.

In vitro transcription and electrotransformation of autonomously replicating RNA

RNA was first transcribed in vitro and then electroporated into BHK-21 cells. To prepare in vitro transcribed template DNA, 6. Mu.g of plasmid DNA was linearized with 4. Mu.L of XbaI (NEB, #R0145) in 80. Mu.L of reaction at 37℃for more than 12 hours. To terminate the cleavage reaction, 4. Mu.L of 0.5M EDTA, 8. Mu.L of 3M sodium acetate and 184. Mu.L of ethanol (ethanol) were added to the reaction. The mixture was thoroughly mixed and frozen at-80℃for at least 20 minutes, and then centrifuged at 13000 Xg at 4℃for 15 minutes to precipitate linearized DNA. After removal of the supernatant, the usually invisible precipitate was carefully washed with 500. Mu.L of 75% ice-cold ethanol and then centrifuged at 13,000Xg for 3 minutes at 4 ℃. The washing process was repeated twice. After complete removal of ethanol, the linearized DNA was resuspended in 10. Mu.L of 10% (v/v) RNAse inhibitor solution (RNasin, promega, #N2111) and immediately used as template for in vitro transcription. According to mMESSAGEUser guidance for the SP6 transcription kit (Thermo, # AM 1340) capping transcription reactions were performed using prepared template DNA. For one 10. Mu.L reaction, 5. Mu.L of 2 XNTP/CAP, 1. Mu.L of 10 Xreaction buffer, 1. Mu.L of GTP,300ng of linear template DNA, 1. Mu.L of mixed enzyme, and 1. Mu.L of RNA-free H ₂ O were used. After incubation at 37 ℃ for 2 hours, the mixture was used immediately or stored at-80 ℃. If necessary, 1. Mu.L TURBO DNase was added to remove DNA, followed by incubation at 37℃for 15 minutes and purification by precipitation with lithium chloride (lithium choloride). For the electroporation step, the inventors modified the procedure ⁸¹ in the previous literature. Briefly, BHK-21 cells cultured in 100 mm dishes were harvested by trypsinization at 90% confluency and then washed twice with 10mL ice-cold DPBS. The 4X 10 ⁶ cells were then suspended in 390. Mu.L ice-cold DPBS and mixed with 10. Mu.L of in vitro transcribed RNA. The mixture was transferred to a frozen 2 mm gap sterile electroporation cuvette and electroporation experiments were performed with three exponentially decaying pulses 3 seconds apart from Bio-rad Gene Pulser II. The device is set as follows: the field strength is 2250V/cm, the capacitance is 25 muF, and the resistance is infinite. Following electroporation, cells were allowed to recover for 10 minutes at 0deg.C, diluted in 10mL BHK-21 cell culture medium, and then transferred to a 100 mm tissue culture dish for normal culture.

Mutant library screening of penultimate amino acids of nsP1 and nsP2

To determine the most favourable combination of penultimate amino acids of nsP1 and nsP2 for host compatibility with RNA, the inventors constructed a two-point site-directed saturation mutant library based on `NNK` (nsP 1: G539X, nsP2: G806X; X represents one of the 20 amino acids encoding a protein). The construction of the mutant library includes two sequential steps: first, an intermediate vector (ML 702|pSP6-nsP1-nsP3-nsP 4-pSG-GFP-P2A-puroR), based on the Sindbis replicon plasmid, was constructed, nsP2 was deleted, and two restriction enzyme sites (NheI and EcoRI) were added. Next, nsP2 was amplified by PCR using degenerate primers and these fragments were ligated to NheI and EcoRI cleaved ML702 by Gibson assembly. The mutant library was then transcribed in vitro and electrotransduced into wild type BHK-21 cells. After transfection, cells were restored for 24 hours, and then 10. Mu.g/mL puromycin was added. The old medium was replaced with fresh puromycin-containing medium every 2 days. Two weeks later, cells were harvested using TRIzol ^TM reagent (Thermo Fisher, # 15596018) to extract RNA. About 3. Mu.g of total RNA was reverse transcribed using HISCRIPT III first strand cDNA synthesis kit (Vazyme, #R312-02) using replicon specific primers. The cDNA was PCR amplified using KOD One ^TM PCR MASTER Mix and target specific primers. The PCR products were sequenced using the rapid NGS service (Tsingke Biotechnology) to quantify the proportion of each amino acid type of nsP1 and nsP2 penultimate amino acids. The mutant libraries were similarly quantified prior to screening. The fold enrichment for each amino acid was quantified as the ratio after screening and before screening. Similar library screening experiments were performed and quantified for replicase-limited BHK-21 stable cell lines, in which nsP4 was replaced with NeoR.

Live cell imaging

During the culture of BHK-21 cells transfected with different versions of Sindbis viral RNA, 100 mm tissue dishes with cells cultured at the indicated time points of the relevant experiments were removed from the incubator and live cell imaging was performed. At the position ofThe bright field and GFP fluorescence of the cells were photographed on FL automated imaging systems (Thermo FISHER SCIENTIFIC) using LPLANFL PH 24 x/0.13 or LPLANFL PH 210 x/0.30 objective lenses. The resulting image was adjusted and cropped using ImageJ2 (https:// ImageJ. Net/software/ImageJ2 /).

Flow analysis

To analyze the expression of fluorescent proteins (i.e., mTagBFP, EGFP, mCherry, and iRFP), single cell suspensions were prepared and flow analyzed on a BD LSRFortessa ^TM cell analyzer. The data were analyzed by MATLAB script (https:// antabilab. Gitub. Io/easyflow /). Unless otherwise indicated, positive cells were determined from non-transfected control cells and Mean Fluorescence Intensity (MFI) was used to represent protein expression levels. To analyze the replication of RNA at early time points of cell entry, replicase levels (i.e., expression levels of nsP 4) were approximated by iRFP levels. Cells with similar replicase levels were classified and MFI of EGFP (expressed from autonomously replicating RNA) per population of cells was calculated as representative of RNA levels. For experiments using flow cytometry to analyze the expression of CD2, the inventors used the method ²⁰ in the previous literature. Briefly, for each repeat, 2. Mu.g of the corresponding plasmid (pEF1α -NLSDCASMINI-NLS-VPR-phU-sgRNA_to_CD2-pEF1α/HTLV-mTagBFP-NLS) was transfected into HEK293T cells using PEI reagent. Three days later, single cell suspensions were prepared, stained with FITC-CD2 antibody (Biolegend, # 309206) in PBS with 5% FBS at 4℃for 1 hour, and analyzed by flow cytometry. The resulting data is processed as described.

Immunofluorescence assay of dsRNA

For immunostaining of double-stranded RNA (dsRNA) of the virus, BHK-21 cells were cultured on 24-well glass plates 24 hours in advance. Cells were fixed with 4% paraformaldehyde (paraformaldehyde) for 20 min at room temperature, washed twice with PBS and permeabilized with 100% ice-cold methanol (methanol) for 30 min at-20 ℃. After washing twice with PBS, cells were blocked with 1% Bovine Serum Albumin (BSA) without IgG for 30 min. Cells were then incubated with 0.5 μg/mL J2 antibody (Scicons # 10010200) containing 1% BSA for 1 hour. Since the diluted J2 antibody may contain a small amount of denatured protein polymer, which may result in a high background for immunofluorescence detection, the diluted J2 antibody needs to be centrifuged at 13000 Xg for 3 minutes before use. After washing twice with 1% BSA, goat anti-mouse IgG H & L (Alexa568 Secondary antibody (Abcam) cells were incubated for 45 min in the dark and then washed twice with PBS in the dark. Then 5. Mu.g/mL Hoechst 33342 was added, the nuclei were stained at room temperature for 10 minutes in the dark, and then washed twice in the dark with PBS. Cells were then imaged on a Dragon Fly200 high speed co-polymerization Jiao Pingtai (ANDOR) with a Plan-Apo 63x/1.4 oil scope (Leica). The image was processed using ImageJ and internal MATLAB scripts. In terms of image processing, the fluorescence Z-axis slice images are first combined, thresholded and segmented according to a maximum value. For dsRNA immunofluorescence images, using grayscale value 200 as the threshold, the particle analysis plug-in ImageJ was applied to the resulting binary image to quantify individual particles. For nuclear images, threshold values were calculated using the Huang method ⁸², and after successive dilation and erosion operations, nuclear masks for individual cells were generated by watershed algorithms. Particle analysis is applied to the nuclear mask image to quantify the size of the nuclei. The output of the particle analysis insert was further processed in MATLAB to estimate the dsRNA quantity per cell in each field of view. By comparing images of control and experimental groups, the true dsRNA signal was empirically defined as particles with at least 6 connected pixels. The signal intensity of individual dsRNA molecules was estimated by using the average intensity of the background-subtracted dsRNA signal of the smallest size. The background-subtracted real dsRNA signals in each field of view are then summed and divided by the estimated intensity of the individual dsRNA and the number of nuclei to yield an estimated number of dsRNA per cell in the target field of view.

RNA sequencing experiments and data analysis

To compare transcriptomes between cells carrying different autonomously replicating RNAs, 30 ten thousand BHK-21 cells carrying repRNA-v3 cells or repRNA-v4 cells (cultured for about 3 weeks after electroporation) were sorted using a BD FACSaria ^TM III cell sorter. Total RNA was extracted using TRIzol ^TM reagent (Thermo FISHER SCIENTIFIC, # 15596018) and library preparation was performed using VAHTS Universal V RNA-seq Library Prep Kit for MGI (Vazyme, # NRM 604-2). The resulting library was next-generation sequenced on the DNBSEQ-T7 platform by An Nuo Yoghurt Gene technologies. The double-ended 150bp sequence with low quality removed was aligned with Mesocricetus auratus genome (GCF_017639785.1_BCM_Maur_2.0_genomic. Fna) with addition of autonomously replicating RNA genome (nsP 1, nsP2, nsP3 and GFP) using STAR 2.7.8a. Genome annotation (gcf_017639785.1_bcm_maur_2.0_genomic.gtf) was downloaded from NCBI and edited to include gene information on autonomously replicating RNA genome. Transcript abundance was calculated from featureCounts and normalized and statistically compared using DESeq 2. RPKM values were then calculated and the bioassay enrichment analysis was performed at Reactome website (https:// reactiome. Org /).

Mutation analysis of autonomously replicating RNA

To analyze mutation rates and mutation spectra of autonomously replicating RNA (repRNA-v 4, unless otherwise indicated) under different conditions, with or without replicase mutagens, and before or after evolution, the corresponding cells were collected and subjected to total RNA extraction and reverse transcription. Sequences of interest (e.g., nsP1-nsP3, EGFP, tetR-VP64, VP64-PadR, dCasMINI, MEK1, KRAS) were amplified from cDNA samples using KOD One ^TM PCR MASTER Mix and target specific primers. Mutation rates and mutation profiles were quantified using NGS sequencing (rapid NGS service for the qing family), and single cDNA variants were obtained by Sanger sequencing of cdnas cloned into plasmids (RUIBIO). For the processing of NGS data, cleaned reads are aligned to corresponding reference sequences using BWA 0.7.17-r 1188. And processing the obtained Bam file by using internal Bash and Python scripts to extract the required mutation information. For analysis of the mutation spectrum, the mutation frequency for each mutation type (e.g., u through c) is calculated as the number of corresponding mutations divided by the total number of sequencing nucleotides. For analysis of the mutations of the evolved RNA, the percentage of mutated nucleotides per nucleotide is expressed.

EGFP evolution experiment

The design of the system and the timelines of the experiment are shown in the top of fig. 37 and 38. The cells used in this experiment were cultured for about 2 weeks after electroporation and were initially used in mutation-related experiments in fig. 26, 28, 31, 32, 36. The evolution experiments were performed in 100mm tissue culture dishes. Cells of 20% confluence were treated with 10. Mu.M Molnupiravir (MCE, # HY-135853) for 2 days and medium was changed daily. Cells were then transferred to 5 100mm dishes and allowed to grow for 3 days. Approximately 3000 ten thousand cells were flow sorted to obtain blue-shifted EGFP mutants, with 0.2% of the cells sorted positive. The sorted cells were incubated with 10. Mu.g/mL puromycin for about 7 days until enough cells were obtained for the second round of evolution, the procedure being the same as the first round. Autonomously replicating RNA (intermediate or final cell population) extracted from cells during the experiment was reverse transcribed and mutations quantified by NGS sequencing. The cDNA library (containing EGFP fragments) was cloned into a vector (pEF1α -EGFP variant-P2A-Puro-BGH polyA-pEF1α/HTLV-iRFP-NLS), transformed into E.coli, and each mutant was identified by Sanger sequencing. To quantify the emission spectra of EGFP variants, 600ng of EGFP wild-type or mutant plasmids were transfected into HEK293T cells in 24 well plates using PEI reagents. After 12 hours of transfection, the fresh medium was replaced and the fluorescence intensities of the EGFP and BFP channels were measured one day later using a flow cytometer.

TetR-VP48 (tTA) evolution experiments

The design of the system and the timeline of the experiment are shown in fig. 42. tetR-VP48 was cloned into a plasmid similar to repRNA-v4 (3 of which the critical amino acids were `VLV` rather than `ALI` in repRNA-v 4), and the resulting autonomously replicating RNA was electroporated into BHK-21 cells expressing nsP4 (stable cell line containing ML700 plasmid). 24 hours after electroporation, the culture medium was replaced with fresh medium containing 5. Mu.g/mL puromycin and 100. Mu.g/mL neomycin (neomycin). After 3 days of selection, a cell population (about 100 ten thousand cells) stably carrying autonomously replicating RNA was obtained and used as starting material for directed evolution experiments in two 100 mm dishes. The steps of mutagenesis and selection are shown in FIG. 42 b. Cells in both dishes were pooled and FACS was performed. The sorted cells were incubated for 3 days with 80. Mu.g/mL puromycin, and collected for RNA extraction, sequencing and mutation analysis. The cDNA library (containing the TetR gene) was cloned into a vector (pEF1α -TetR_mutant-VP48-BGH polyA-pEF1α/HTLV-iRFP-NLS), transformed into E.coli, and individual mutants were determined by Sanger sequencing. To quantify ligand sensitivity of TetR variants, 400ng of TetR (wild-type or mutant) -VP48 plasmid and 200ng of reporter plasmid (pTRE 3G-Citline-NLS) were co-transfected into HEK293T cells in wells of 24-well plates containing 1ng/μl of doxycycline (Dox) medium. After 12 hours of transfection, the medium was replaced with fresh medium containing Dox and the fluorescence intensity was measured one day later with a flow cytometer.

VP64-PadR evolution experiment

The design of the system and the timeline of the experiment are shown in the top of fig. 45. VP64-PadR ⁵⁶ (ENCU, given by the professor She Haifeng) was cloned into the repRNA-v4 plasmid and the resulting autonomously replicating RNA was electroporated into a stable cell line expressing nsP4 (containing the ML700 plasmid) and BHK-21 cells carrying a stably integrated reporter plasmid ((O _PadR)₆ -PHCMVMINI-PuroR-NLS-mCherry-hPEST-BGHpolyA-pEF1α/HTLV-tag BFP 2-NLS), 24 hours after electroporation, replaced with fresh medium containing 5. Mu.g/mL puromycin and 10. Mu.g/mL blasticidin (blasticidin), after 3 days of selection, a population of cells (about 500 ten thousand cells) stably carrying autonomously replicating RNA was obtained and served as starting material for a directed evolution experiment in a 100mm dish, which involved two independent directed evolution activities, the corresponding mutation and selection procedure is summarized in the bottom of FIG. 45, FIG. 49. On day 21, all cells from the first experiment and cells from the second experiment screened by mCherry were collected for RNA extraction, sequencing and mutation analysis. CDNA libraries (containing PadR genes) were ligated to one vector (pEF1α -VP64-PadR _mutant-BGHpolyA-pEF1α/HTLViRFP-NLS) and transformed into E.coli, single mutants were determined by Sanger sequencing to quantify the ligand sensitivity of the PadR variants, 400ng of the VP64-PadR (wild-type or mutant) plasmid and 200ng of the reporter plasmid ((O _PadR)₆ -PHCMVMINI-Puro-NLS-mCherry-hPEST-BGHpolyA-pEF1α/HTLV-mTagBFP 2-NLS) were co-transfected into HEK293T cells in one well of 24-well plates in a medium well containing Sodium Ferulate (SF).

CasMINI evolution experiment

The design of the system and the timelines of the experiment are shown in fig. 67 and 68. NLS-dCasMINI (v 4) -NLS-VPR ²⁰ was cloned into the repRNA-v4 plasmid and the resulting autonomously replicating RNA was electroporated into BHK-21 cells expressing nsP4 (a stable cell line containing the ML700 plasmid) and carrying a stably integrated reporter plasmid (pTRE 3G-PuroR-P2A-NLS-mCherry-BGHpolyA-phU6-sgRNA_TetO-pEF1α/HTLV-tagBFP-NLS). 24 hours after electroporation, the old medium was replaced with fresh medium containing 5. Mu.g/mL puromycin and 10. Mu.g/mL blasticidin. After 5 days of selection, a cell population (about 200 ten thousand cells) stably carrying autonomously replicating RNA was obtained and used as starting material for directed evolution experiments, performed in 100mm dishes. On day 22, cells were collected for RNA extraction, sequencing and mutation analysis. The cDNA library (containing dCasMIN genes) was ligated into a vector (pEF1α -NLS-dCasMINI _mutant-NLS-VPR-BGH polyA-pEF1α/HTLV-iRFP-NLS), transformed into E.coli and mutant identified by Sanger sequencing. The inventors obtained only one enriched mutation in the two replicates (S246R). The inventors characterized the three functions of the mutant, including transcriptional activation, base editing and nuclease functions. The sgRNA information and corresponding target gene information were from previous studies ²⁰. As a transcriptional activator, the inventors characterized the ability of the wild type (dCasMINI (V4)) and mutant (S246R) to activate the reporter system and endogenous gene CD2 used in the evolution process. As a base editor or nuclease, the inventors tested two endogenous genes ifnγ and VEGFA. The plasmid information used in these experiments is detailed in Supplementary Table. In all assays, 2 μg of plasmid was transfected into HEK293T cells in one well of a 12-well plate. 12 hours after transfection, replaced with fresh medium, and after 60 hours, the cells were collected for flow analysis or sequencing.

MEK1 evolution experiments

The design of the system and the time line of the experiment are shown in fig. 56. The human MEK1 cDNA was cloned into repRNA-v4 plasmid and the resulting autonomously replicating RNA was electroporated into nsP4 expressing BHK-21 cells (stable cell line containing ML700 plasmid). 24 hours after electroporation (i.e., day 1), the medium was replaced with fresh medium containing 5. Mu.g/mL puromycin. After 4 days of selection (i.e., day 5), molnupiravir of 2. Mu.M was added to the medium. On day 7, the therapeutic drug (i.e., 2. Mu.M cobicitinib, 5. Mu.M trametinib (trametinib), or 50. Mu.M semetinib (selumetinib)) was added to the medium. After one week (i.e., day 14), cells were collected for total RNA extraction, sequencing, and mutation analysis. The cDNA library (containing the MEK1 gene) was cloned into a vector (pEF1α -MEK1_mutant-BGHpolyA-pEF1α/HTLV-iRFP-NLS), transformed into E.coli, and individual mutants were identified by Sanger sequencing. To quantify the drug sensitivity of MEK1 variants, 400ng of a MEK1 (wild type or mutant) containing plasmid and 200ng of an SRE reporter plasmid (7X SREPHCMVMINI-NLS-mCherry-BGHpolyA-pEF1α/HTLV-mTagBF P-NLS) were co-transfected into HEK293T cells on 24 well plates, the medium containing 2. Mu.M cobratinib. After 12 hours of transfection, the medium was replaced with fresh medium containing cobicitinib, and after one day the fluorescence intensity was measured with a flow cytometer.

KRAS (S17N) evolution experiment

The design of the system and the time line of the experiment are shown in fig. 61. The cDNA of wild-type KRAS or KRAS (S17N) of human origin was cloned into the repRNA-v4 plasmid and the resulting autonomously replicating RNA was electroporated into BHK-21 cells expressing nsP4 (stable cell line containing ML700 plasmid). 24 hours after electroporation (i.e., day 1), the medium was replaced with fresh medium containing 5. Mu.g/mL puromycin. After 4 days of selection (i.e., day 5), molnupiravir of 2. Mu.M was added to the medium. After 10 days of culture, the cells were collected for total RNA extraction, sequencing and mutation analysis. The cDNA library (containing the KRAS gene) was cloned into a vector (pEF1α -KRAS_mutant-BGHpolyA-pEF1α/HTLViRFP-NLS), transformed into E.coli, and single mutant identified by Sanger sequencing. To quantify the signal activity of KRAS variants, 400ng of KRAS (wild type or mutant) containing plasmid and 200ng of SRE reporter plasmid (7 XSRE-PHCMVMINI-NLS-mCherry-BGHpolyA-pEF1α/HTLV-mTagBF P-NLS) were co-transfected into HEK293T cells in 24 well plates. After 12 hours of transfection, the medium was replaced with fresh medium and after one day the cell number and fluorescence intensity were measured with a flow cytometer.

Examples

Example 1 engineering autonomously replicating RNA suitable for in vivo evolution

RNA replicons derived from alphaviruses (positive strand RNA viruses) are widely used not only for expression of heterologous proteins, such as vaccine antigens ^37–39 in animal cells and tissues, but also for mammalian synthetic biology ^9,40. However, they induce a strong cytopathic effect ^35,41, disrupt the physiological function of the cells, and prevent long-term replicon proliferation. Thus, host cell compatibility must be improved by engineering an autonomous replication of the RNA by the alphavirus, thereby utilizing such RNA for directed evolution in vivo.

Based on the work of the Sindbis virus (one of the alphaviruses) derivative replicons, the inventors began ^42,43 with two replicon versions that proved to have low cytopathic properties. The first version (repRNA-v 1) contains a mutation of nonstructural protein 2 (nsP 2), while the second version (repRNA-v 2) contains this mutation and two mutations at the linker peptide of nsP1-nsP2 and nsP2-nsP3 (i.e., the penultimate residues of nsP1 and nsP 2; fig. 1), respectively. These replicons were transcribed in vitro and electrotransformed into BHK-21 (little hamster kidney fibroblasts) cells (see the methods section for details). The inventors found that the number of cell survival after electrotransformation was significantly higher for the second version compared to the first version (fig. 2), indicating that perturbation of the treatment with multimeric proteins (i.e. P123 or P1234) greatly affected the cytopathic condition ^42,43. Thus, the inventors have screened for these two linking residues (FIG. 3, see methods section). The inventors found that alanine was highly enriched at the penultimate residue of nsP1, while no amino acids were significantly enriched at nsP2 (fig. 3 and 4), suggesting that a single linker mutation of repRNA-v2 could improve cell compatibility. Furthermore, since the exact same mutation of the penultimate residue of nsP1 was demonstrated to interfere with viral replication ⁴², it can be reasonably deduced: compatibility of host cells may be improved by interfering with RNA replication.

Based on the above findings, the present inventors have attempted to further improve host cell compatibility by expressing the RNA replicase nsP4 in the host genome (rather than from the RNA itself). This will restrict RNA replication, as replication reactions will be limited by replicase enzymes and non-self-catalyzed. The inventors then integrated a constitutively expressed nsP4 into the genome (see methods for details) and re-pooled the same residue positions using RNAs without nsP4 (fig. 5a and b). Interestingly, the cells in this screen grew faster than the screen using repRNA-v2 (FIG. 6). Furthermore, alanine was again enriched in the nsP1-nsP2 linker, as in the previous screen, again without significant enrichment of amino acids in the other ligation position (fig. 7).

Based on these screens, the inventors constructed two new versions of autonomously replicating RNA: repRNA-v3, which contains alanine and isoleucine as penultimate residues of nsP1 and nsP2, respectively, based on repRNA-v 2; repRNA-v4 then contains the same mutation as repRNA-v3, but no nsP4 (thus requiring replication in cells expressing nsP 4) was present (when two autonomously replicating RNA versions (repRNA-v 3 and repRNA-v 4) were constructed, the inventors tried to compare the same autonomously replicating RNA with replicases expressed from different positions, whether from the RNA itself (repRNA-v 3) or from the host genome (repRNA-v 4)), thus, it was desirable that both versions of RNA include the same penultimate residue of nsP1 and nsP 2. Since alanine was enriched in both screens of nsP1-2 adaptor residues, the inventors selected alanine as the second residue of nsP1 in both versions, in contrast, no amino acid was significantly enriched in the nsP2-3 adaptor from either screen, thus, the inventors decided to select such an amino acid (the penultimate residue for nsP 2) that could be enriched in one of the two versions of nsP2, when replicases expressed by the host were used, because of which is likely to have improved in vivo, and thus the invention has evolved because of the repeated selection of one of the penultimate residues. The inventors compared these two new versions to repRNA-v 2. The inventors found that the linker peptide mutations did improve cell compatibility (i.e., repRNA-v3 versus repRNA-v 2) and that restricting the level of replicase by expression from the host genome greatly enhanced host cell compatibility (i.e., repRNA-v4 versus repRNA-v 3) (fig. 8 and 9). These results indicate that viral RNA replication appears to be acceptable to the host cells when replicase levels are limited, enabling the production of 10 ⁸ host cells carrying autonomously replicating RNA within a week (fig. 9). However, the above measurement is performed one week after electroporation, so the acceptance of RNA by host cells in early and late phases (i.e., short-term and long-term stability) is still unknown.

Example 2-engineered RNA has both short-term and Long-term stability

The inventors first quantified the short term stability of three autonomously replicating RNA versions (repRNA-v 2 to v 4) by measuring EGFP expressed by RNA at early time points of entry into cells (fig. 10 and 11). For the version without restriction replicase, the inventors found that the optimization of the linker peptide (repRNA-v 3) improved host cell compatibility over the non-optimized version (repRNA-v 2), resulting in a higher maximum ratio of EGFP-positive cells and a higher proportion of EGFP-positive cells remaining at 72 hours (FIG. 12). However, for both RNAs, the proportion of EGFP-positive cells dropped below 10% within 3 days after electroporation, indicating overall lower host compatibility. In contrast, for the replicase limited version (repRNA-v 4), the EGFP positive cell proportion could be maintained up to about 50% on day 3 and showed a pattern of behavior dependent on replicase levels (fig. 13). Thus, the replication pattern of the limited replicase may achieve lower cytopathology to better maintain continuous RNA replication. From a mechanistic point of view, host transcriptome analysis showed that replication without replicase restriction (i.e., repRNA-v 3) significantly upregulated genes associated with the interferon signaling pathway (FIG. 14), a reflection of known virus-induced cytopathogenicity ⁴⁴.

Short term stability of RNA showed an increasing trend with decreasing replicase levels, as the EGFP positive proportion showed a more stable maintenance state from EGFP levels to steady state (36 hours) to end (72 hours) (fig. 13, left side of fig. 15). To test whether reduction in replicase levels has a causal relationship with improvement in short term stability, the inventors created a new host cell line (referred to as a "low replicase host") by inserting a stop codon in front of nsP4 (fig. 16). Replicase levels are further reduced compared to previous host cell lines (referred to as "high replicase hosts"). In this new host cell, EGFP levels expressed from RNA (repRNA-v 4) were as expected to be lower than in the previous host cell (compare FIG. 17 and FIG. 13). Most importantly, the short-term stability of autonomously replicating RNA was indeed enhanced in the new host cell (fig. 15). Together, these data indicate that a decrease in replicase levels results in a decrease in RNA replication rate (i.e., a decrease in EGFP levels), resulting in an increase in short-term stability of autonomously replicating RNA.

Based on these data, the inventors constructed a simple model of RNA replication kinetics, suggesting that steady-state RNA levels are proportional to replicase levels. By quantifying the steady-state RNA (EGFP) levels at different replicase levels in the two host cell lines, the inventors have found that for low replicase levels, steady-state RNA levels are linearly proportional to replicase levels (fig. 18). However, for high replicase levels (with high replicase hosts), it appears that steady-state RNA levels reach a plateau after a linear phase (fig. 18), which may reflect that the extent of RNA loading by the host cells is determined by RNA-induced cytopathological effects. In agreement with this, when cultured for longer periods, only cells with low replicase levels in the high replicase hosts survived (FIG. 19), which is significantly different from the low replicase hosts (FIG. 20).

In summary, these quantitative data help describe the process of maintenance of RNA replication over a short period of time and illustrate the kinetics and function of the limited replicase pattern of RNA replication. Kinetically, the limited replicase mode operates in a saturated kinetic state with its steady state level regulated, helping to improve host compatibility. Functionally, this pattern achieves the production of 10 ⁸ cells (starting from 4×10 ⁶ cells) within a week by enhancing host compatibility (fig. 9). Importantly, RNA (repRNA-v 4) was stably propagated for more than one month (fig. 21, fig. 22), from which RNA was replicated uniformly in single cells, as confirmed by immunostaining of replication intermediate double-stranded RNA (dsRNA) (fig. 23, fig. 24, see methods for details). By further quantifying the level of viral genome using RNA sequencing (see methods for details), the inventors estimated that each cell can carry about 100 RNA molecules (FIG. 25, the inventors used bulk poly-A RNA-seq to quantify the expression levels of autonomously replicating RNA and endogenous genes. The inventors found that repRNA-v4 (in high replicase hosts) were expressed at about 60% of the Gapdh gene, as measured by reading maps to the nsP1-3 reads. Since GAPDH MRNA in mammalian cells is typically 1000 molecules per cell ³, the inventors inferred that the engineered autonomously replicating RNA, repRNA-v4, has at least on the order of 100 molecules per cell. Thus, up to more than 100 billion RNA molecules (10 ⁸ cells. Times.100 copies/cell) can be subsequently diversified and selected within a living mammalian cell.

Example 3-controlled RNA diversification by intervention with RNA replication accuracy

The inventors next explored the mutagenic capacity of this engineered system, which can determine the evolution and adaptation efficiency of autonomously replicating RNA. The inventors first quantified the basal mutagenesis rate of RNA and then tried to regulate this rate by intervening in the accuracy of replicase.

To quantify the fidelity of replicase, the inventors used second generation sequencing to measure the mutation rate of RNA after 1,3, 7, 14 and 21 days of maintenance in cells (a and b of fig. 26). To establish a baseline, the inventors sequenced plasmid DNA as well as in vitro transcribed RNA and found that sequencing and in vitro transcription together could introduce a mutation rate of about 0.4 bases/kb total. Once in the cell, mutations on the RNA accumulated at a rate of about 0.01 bases/kb per day, reaching about 0.22 bases/kb after 21 days (b of fig. 26, fig. 27), which contained four major mutation types (i.e., a to U, A to G, G to a and C to U) (fig. 28). Thus, replicases do not introduce many errors during long-term RNA replication, consistent with slow replication of RNA. It is critical that such low basal mutation rates provide opportunities for a controlled mutagenesis rate.

To alter the fidelity of replicase, the inventors measured the mutation rate ⁴⁵ of RNA in host cells carrying mutant nsP4, which is known to decrease accuracy. The inventors observed only a slight but statistically significant increase in mutation rates for the four mutation types, which, however, only very limited improved the diversity capacity of the RNAs (fig. 29). The inventors next studied small molecule mediated RNA replicase intervention, as some therapeutic nucleoside analogs combat RNA viruses (e.g., molnupiravir for treatment COVID-19) ^46–50 by reducing the fidelity of RNA replicase. The present inventors tested four different nucleoside analogs and compared their ability to eliminate the EGFP signal expressed by RNA, with analogs having greater ability to induce mutations resulting in faster accumulation of non-fluorescent EGFP mutants (FIG. 30, FIG. 31). The inventors found that favipiravir and molnupiravir did not affect cell growth among these four drugs (fig. 70) and were effective in reducing EGFP signal dose-dependently (fig. 32) when added to the culture medium of host cells containing autonomously replicating RNA in a dose-dependent manner within 3 days (fig. 31, fig. 33). By quantifying the rate of RNA mutation over the course of time of drug treatment, the inventors have found that both drugs can increase the mutagenic capacity of RNA replicases by two orders of magnitude, achieving mutation rates of up to 1 base/kb per day (FIG. 34), which should enable rapid diversification of RNA-based synthesis devices. Importantly, the enhanced mutagenesis rate of the orthogonal RNA replication system is likely not to affect the genes expressed by the host genome, as can be seen from the stability of the iRFP signal (expressed with nsP 4) and the EGFP signal in the control cell line (fig. 35).

The inventors further compared the mutation profiles of RNAs with nucleoside analogs or vector controls. The inventors found that molnupiravir, but not favipiravir, induced a relatively balanced mutation spectrum by increasing the mutation frequencies of all four major transition types on average (fig. 36). Thus, the inventors have chosen to use molnupiravir, which can induce a relatively broad and balanced mutation profile and allow the inventors to achieve diversification of RNA in a controlled manner.

Example 4-blue-shifting of EGFP Signal based on REPLACE System

By correlating the products of autonomously replicating RNAs with phenotypes, these RNAs can in principle be mutated to create diverse phenotypes that can be targeted for selection. Thus, the inventors explored the utility of orthogonal RNA replication systems by REPLACE. By replacing, genes containing the gene sequence of the protein of interest can be expressed by subgenomic promoters on RNAs that can be replicated, mutagenized and targeted for selection.

The inventors have attempted to test replacement by color change of EGFP signal, since EGFP itself is a material that validates directed evolution tools, since GFP would be changed to BFP ^51,52,19 by only one amino acid substitution. Specifically, the inventors placed the EGFP gene under the control of a subgenomic promoter that autonomously replicates RNA (FIG. 37), and then cultured a cell population containing such RNA (see methods for details). Then molnupiravir was added to the medium to accelerate mutagenesis, which was continued for two days (FIG. 38). Thereafter, flow analysis of the GFP and BFP signals showed that a small fraction of the cells were off diagonal, indicating that the fluorescence was blue shifted (middle of fig. 38). By cell sorting, the inventors directed to selecting two cell populations exhibiting different blue shift signals, which were subjected to a second round of mutagenesis and screening. By sequencing, the inventors observed that one highly enriched missense mutation (r.196U > C, i.e. mutation of the 196 th nucleotide of mRNA from U to C) was found in the vertical population, while another enriched missense mutation (r.608C > U) was found in the off-diagonal population (fig. 39, fig. 40). These two enriched mutations were associated with the conversion of GFP to BFP or to saphire ^53,54 signal, respectively ⁵¹ (fig. 41). Notably, although two mutants that exhibit a blue shift signal each require mutation of only one residue, the two residues resulting in two mutants are far apart and the two mutants are easily detected after the first sorting, demonstrating the feasibility of the REPLACE system in evolving synthetic devices that require multiple mutations.

Example 5 engineering of transcriptional regulatory proteins Using the REPLACE System

The inventors next attempted to utilize REPLACE to evolve synthetic transcriptional regulatory proteins, including ligand-controlled Transcription Factors (TF) and Cas (CRISPR-associated) protein-based transcriptional regulatory proteins, which have been widely used ⁷ in mammalian synthesis circuits.

The inventors first constructed synthetic TFs that could reduce or enhance ligand sensitivity. As a test of the REPLACE system, the inventors' ideal approach is to choose to reproduce the research results ^16,18 that have been achieved by other directed evolution methods: tetR was evolved to reduce its sensitivity to doxycycline (doxycycline). Briefly, autonomously replicating RNA (i.e., tTA) carrying TetR-VP48 was continuously diversified for 7 days in a relatively low dose molnupiravir (2. Mu.M) environment (FIGS. 42, a and b, see methods for details). Meanwhile, the concentration of doxycycline and puromycin (puromycin) in the medium gradually increased over a period of 2 weeks, during which the TetR mutant, which was able to bind to DNA (by increasing the concentration of doxycycline resistance) and drive the expression of puromycin resistance gene, would be enriched. The inventors then selected cells expressing high levels of mCherry signal and extracted their RNA for reverse transcription and sequencing. It is appreciated that the inventors found three enriched missense mutations (> 5%) with the highest enriched mutation T103P located directly in the ligand binding pocket and the other two mutations (R80C and E157G) located near the pocket ⁵⁵ (fig. 43). Using the reporter system of HEK293T cells, the inventors found that the TetR variant (determined from Sanger sequencing) increased in resistance to doxycycline (i.e. decreased sensitivity) as the number of mutated residues increased from one to three (fig. 44). Taken together, these results indicate that replacement can construct new synthetic TF through autonomous replication RNA diversification and directed selection within mammalian cells.

The inventors next attempted to engineer a newly developed ligand-controlled mammalian synthetic TF, VP64-PadR ⁵⁶, whose DNA binding capacity can be turned off by U.S. FDA approved drugs (sodium ferulate ). The purpose of the engineering is to reduce its ligand sensitivity or to increase the dynamic range of its response. To reduce ligand sensitivity, the inventors directed evolution of RNA containing VP64-PadR mutants by RNA diversification followed by selection with increasing puromycin concentration in the presence of ligand (sodium ferulate) (FIG. 45, detailed methods). By reverse transcription and sequencing of RNA extracted from the surviving cell population, the inventors determined 8 enriched missense mutations (fig. 46), 4 of which (E28K, E92G, G153S, K E) were located in the vicinity of the ligand binding pocket ⁵⁷. The inventors then quantified ligand sensitivity of the first generation sequencing-derived mutants by a reporter system and found that mutants with single (E92G or G153S) or double (E28K/G153S) mutations near the binding pocket exhibited reduced sensitivity (fig. 47). Unexpectedly, mutations outside the binding pocket, either by themselves or in conjunction with mutations near the binding pocket, can reduce (FIG. 47) or even reverse (FIG. 48) the sensitivity to sodium ferulate (T55I/Q78P, L P/K166E, E K/T55I/Q78P), which means that ligand binding has potential allosteric regulation and cooperative interactions between the two classes of residues.

Next, the inventors tried to expand the response dynamic range of VP64-PadR by constructing mutants with enhanced activation in the absence of ligand. The present inventors first performed RNA diversification and then selected RNAs with enhanced activation of target genes without adding sodium ferulate (fig. 49, see methods for details). Finally the inventors confirmed 6 enriched missense mutations, three of which (Y25C, E92K, D G) were located near the binding pocket (fig. 50). Notably, padR variants containing mutations at residues near one or more of the binding pockets (Y25C, Y C/S174N, D G/S174N, Y C/E92K, D V/R86K/D130G/S174N) will also exhibit enhanced target activation in the absence of ligand. However, the dynamic range of the reaction (quantified by fold change in target reaction with or without ligand) was reduced compared to the wild type (fig. 51). In contrast, the dynamic range of the response was at least comparable to that of the wild type for mutants carrying the binding pocket away (D74V, D G/D74V/M178T). Importantly, one of the variants (D21G/D74V/M178T) showed a dramatically enhanced dynamic range (85-fold) compared to the wild type (38-fold) (fig. 51), while ligand affinity was only slightly altered (fig. 52).

After establishing that replacement has the ability to engineer ligand-controlled mammalian synthesis of TF, the inventors further sought to evolve Cas-based transcriptional regulatory proteins. Miniaturized Cas12f protein CasMINI ²⁰, about 500 residues in length, can be conveniently delivered into cells, previously engineered according to rational design principles ²⁰. The inventors hoped to further increase the transcriptional regulatory capacity of this protein by replacing. The inventors have diversified and screened autonomously replicating RNAs carrying endonuclease-inactivated CasMINI-v4 ²⁰ (dCasMINI-VPR) (FIGS. 67 and 68). Surprisingly, in two independent experiments, the inventors confirmed only one enriched mutation (S246R) (fig. 69). By quantifying transcriptional activation capacity by a reporter system, the inventors found that the S246R variant had about 30% improved activation capacity for the TetO-containing promoter (used in the evolution experiments) compared to the parent (fig. 53), and about 20% improved activation of an endogenous gene (CD 2) with an sgRNA (fig. 54). The inventors further found that the evolved mutants did not result in a significant enhancement of the relevant base editing ability and nuclease activity (fig. 55), probably because the transcriptional regulatory activity may not be correlated with the activity of base editing or DNA cleavage ²⁰.

Together, these results demonstrate the ability and versatility of replacement in constructing ligand-controlled or Cas-based transcriptional regulatory proteins synthesized by mammals, demonstrating that this system has the ability to improve other TF in mammalian synthetic biology applications.

Example 6-autonomous replication of RNA carrying MEK1 to achieve Darwin-type adaptation

By virtue of the orthogonality of the RNA replication system, the REPLACE system enables a continuous mode of operation-RNA can be autonomously replicated across generations, opening up a way for the cell to be constructed in a darwinian-like manner (i.e. by adaptive selection). Thus, the present inventors have next tested whether mammalian cells can utilize REPLACE to evolve genes of great physiological significance, thereby autonomously adapting to new environments.

The inventors first sought whether cells carrying autonomously replicating RNA of the human MEK1 gene could adapt to the environment containing the MEK1 allosteric inhibitor. Since it has been confirmed that in vivo or in vitro mutagenesis of MEK1 can produce the anti-inhibitor mutant ^58,19 of MEK1, in principle, cells can survive in the presence of inhibitors using the evolutionability of autonomously replicating RNA. The inventors then used repRNA-v4 containing the wild-type MEK1 gene expressed from the subgenomic promoter to transfect cells (day 0) (FIG. 56, see methods for details). Molnupiravir was added to the culture starting on day 5 to diversify the RNA. Then starting on day 7, survival pressure was applied to the cells with three independent MEK1 allosteric inhibitors and cells were collected for sequencing on day 14. The diversification and selection of RNA was performed simultaneously in the inhibitor environment over a period of 7 days, enabling adaptation of darwinian style (fig. 56).

Interestingly, the inventors found that surviving cells contained RNA highly enriched for MEK1 mutations (i.e., >5% in one or more biological replicates) and that several mutations were enriched in all three inhibitors (fig. 57, fig. 58), suggesting that darwinian adaptation to allosteric inhibitors of MEK1 could occur through a conserved mechanism. Structurally, mutations are located within different functional domains ^59,58,60 (fig. 59): the inhibitor binding pocket (L115P, V211D, L P), the negative regulatory domain (R47Q, Q56P, K N), residues that may interfere with inhibitor function (H119Y) and nuclear export signal (E39A) ⁶¹. Many of which were previously found in the mutagenic screen ⁵⁸ for MEK 1. Sanger sequencing enabled the inventors to identify each MEK1 mutant, which on average carries 1 to 3 mutations.

The inventors next used a fluorescence reporting system to measure MEK1 activity (i.e., SRE reporting system ^19,62 reflecting MAPK/ERK signaling activity) to confirm the resistance of the different mutants to cobicitinib (Cobimetinib). The inventors found that all mutants, except the H119Y mutation, showed stronger reporter activity (i.e. enhanced inhibitor resistance) than the wild type in the presence of the inhibitor (fig. 60). Surprisingly, the basal signaling activity exhibited when two mutants were contained in the inhibitor binding pocket (L115P/V211D) was greatly reduced compared to the single mutant (L115P and V211D), indicating interactions between residues in the allosteric pocket (fig. 60). It was found that the combined mutation of the inhibitor binding pocket and the negative regulatory domain (Q56P/V211D and R47Q/K57N/V211D) dramatically increased the activity of MEK1 in the presence or absence of the inhibitor compared to the binding pocket-only mutant (FIG. 60). For one of the mutants (Q56P/V211D), the inhibitor even increased its activity (rather than inhibition) (fig. 60). These results underscore the ability of REPLACE to traverse complex sequence functional maps of MEK1 during inhibition of cell adaptation to MEK 1.

Example 7 escape of dominant negative KRAS Gene Using the REPLACE System

After determining that replacement can adapt cells to external challenges (i.e., small molecule inhibitors), the inventors next began to investigate whether and how cells equipped with autonomously replicating RNA adapt to the challenges inherent in cells. The present inventors selected a protooncogene, KRAS, which is one of the most frequent mutant genes ⁶³ in cancer. In addition to small molecule-based cancer KRAS targeted therapies, researchers have proposed and tested dominant negative gene therapies ^64,65. The oncogenic KRAS activity (and cell proliferation) ⁶⁶ is inhibited by introducing a dominant negative KRAS allele (S17N). In principle, cells can develop resistance to this treatment by reversing a single mutation (S17N), however there are other potential ways to confer the same resistance to cells.

Thus, the inventors sought to investigate whether and how cells carrying autonomous replication RNAs of the KRAS (S17N) allele overcome their (intracellular) inhibition of KRAS activity by darwinian adaptation (fig. 61). As a control, the inventors also evolved cells carrying autonomously replicating RNA of wild-type KRAS (fig. 61). RNA was first continuously diversified in cell culture medium containing molnupiravir for 9 days (FIG. 61, detailed methods), during which time cells with increased KRAS activity and enhanced proliferation should have greater survival advantage than cells with inhibited KRAS activity. By sequencing the RNA of the surviving population, the inventors found enriched missense mutations on KRAS (S17N) that were absent on six wild-type KRAS (fig. 62, fig. 63). Interestingly, the present inventors found that an enriched mutation changed asparagine (asparagine) N17 to a hydroxy amino acid threonine (threonine, N17T) instead of serine (N17S) that was changed in the wild type (fig. 62). Since serine or threonine are both critical for guanine nucleotide binding—they can be ion coordinated ⁶⁷ with Mg ²⁺ through hydroxyl groups, the N17T mutation may be sufficient to reverse the impact ⁶⁸ of the dominant negative phenotype of KRAS (S17N). While several other mutations are located in important functional regions ⁶⁹ (fig. 64), including switch I region (Y32C) ⁷⁰ responsible for effector participation and hydrophobic hinge (L79F) ⁷¹ mediating critical allosteric interactions. However, it is still difficult to determine which mutation is functionally related to KRAS that is free of dominant negatives.

Thus, the inventors used the SRE reporting system to quantify the activity of each KRAS mutant (comprising up to four mutations) while characterizing their effect on cell proliferation. As a control, the inventors found that the signal activity of wild-type KRAS was significantly enhanced compared to the dominant negative mutant (S17N), whereas the well-known oncogenic KRAS mutation, G12C, also resulted in higher activity (top of fig. 65). Consistent with the activity readings, both wild-type KRAS and KRAS (G12C) induced faster cell proliferation compared to KRAS (S17N), whereas both variants were not themselves distinct in terms of cell proliferation (bottom of fig. 65). The inventors then tested the confirmed mutants evolved from KRAS (S17N) and found that the N17T mutant reversed the dominant negative effect as it resulted in significantly higher levels of activity than the wild type, and comparable cell proliferation rates to the wild type (figure 65). Another mutation, L79F, further enhanced signaling activity, but did not enhance cell proliferation (FIG. 65). These results indicate that T17 is better ion coordinated with Mg ²⁺ than S17 of wild-type KRAS, whereas higher signaling activity may not lead to an enhancement of cell proliferation. For other mutants without N17T mutation, none resulted in a considerable proliferation advantage, although they all gave statistically enhanced KRAS signaling activity (fig. 66), meaning that these mutants might confer survival advantages to other cell types during the period of escape from dominant negative inhibition.

Taken together, these results demonstrate that cells are able to escape the effects of dominant negative KRAS by replacement-mediated darwinian adaptation.

Example 8-discussion

Synthetic biology is often compared to electrical engineering and like disciplines ⁷² because they all use engineering principles to construct pathways and systems that operate as designed. However, biological systems are complex in nature and evolve naturally, requiring the theory and tools of synthetic biology beyond the scope of traditional disciplines to achieve evolutionability ²⁵ of the constructed biological systems. To achieve this goal, the inventors developed REPLACE: RNA replicates orthogonally in mammalian cells, continuously evolves and stably propagates. Not only can the replacement of biomacromolecule engineering compatible with mammals be directly realized in a mammalian system, but also synthetic evolutionary properties can be provided for mammalian cells, so that the engineering cells can adapt to new environments by utilizing the evolutionary properties of autonomous replication RNAs.

One key concept and technological advance in comparison to existing directed evolution methods is the tuning and design of RNA virus replication systems to accommodate directed evolution in vivo in mammalian cells. Although viruses have previously assisted directed evolution ^73,74,16,18 of biological macromolecules, replacement is fundamentally different because only a portion of the viral genome is propagated entirely within the cell and does not produce live viruses. In contrast to RNA viruses that accumulate the common mutation ^75,76 during replication (e.g., SARS-CoV-2), sindbis virus-derived RNA in the REPLACE system replicates under limited patterns and saturation kinetics of replicases to ensure low levels of replication and underlying error accumulation, which lays a foundation for chemically controlling the accuracy of RNA replicases (and thus RNA mutagenesis). In contrast to natural RNA viruses, the mutations designed by the present inventors to autonomously replicate RNA are mainly from perturbation of RNA replicase enzymes, rather than from steady state (undisturbed) replication. This finding also suggests the widespread use of mutagenic nucleoside analogues to treat the potential adverse effects ^49,77 of COVID-19, a double-edged sword, which may greatly accelerate the emergence of new viral variants by inducing mutations and promoting rapid evolution of the virus.

The versatility of the REPLACE system enables the inventors to use directional selection to engineer functional biomolecules, including synthetic transcriptional regulator proteins, and to enable darwinian adaptation of cells in the face of challenge. These successful applications highlight three significant features of REPLACE. First, the inventors' RNA-based evolutionary system is largely orthogonal to the host cell and uniquely enables in vivo evolution of RNA devices within the host cell (unlike in vitro evolution ^78,79). Each cell can hold about 100 copies, creating a huge library of mutants to ensure a wide variety of evolutionary experiments. Second, the present inventors' system provides flexibility for diversification and selection steps—diversification not only can confer a relatively broad mutation spectrum, but is also fully controllable; the selection may be directional or adaptive based; these two steps can even be performed simultaneously to achieve continuous evolution. Third, devices based on autonomously replicating RNA provide a simple and powerful means to enable mammalian cells to accomplish adaptation, which is a complex and adaptive task that is difficult to achieve with conventional synthetic biology tools.

The inventors contemplate that replacement has a wide range of applications including mammalian biomacromolecule engineering, synthetic biology, and developmental biology (e.g., for tracking cell lines). To further expand its range of applications, it may be improved in several ways, including screening methods (e.g., incorporating negative selection), target patterns (e.g., evolving functional RNAs), and cell compatibility (e.g., further reducing cytopathology and expansion to other cell types).

Broadly, the adaptive RNAs developed by the present inventors are evolutionary, which will facilitate the engineering and use of mammalian RNA synthesis devices to form new models. The hot trend ^32,33,80 of current RNA-based therapies may promote the development of future evolutionary and adaptive RNA-based therapeutic devices that can be delivered directly into cells of living animal tissue to address unforeseen challenges.

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Although the present application has been illustrated and described with respect to exemplary embodiments thereof, it will be understood by those skilled in the art that the foregoing embodiments are not to be construed as limiting the application in any way, and that changes, substitutions and alterations can be made hereto without departing from the spirit, principles and scope of the present application as defined by the appended claims.

Claims

1. An RNA replicase-assisted continuous in vivo evolution (REPLACE) system comprising autonomously replicating RNA and a host cell,

Wherein the autonomously replicating RNA is repRNA-v4,

Wherein the autonomously replicating RNA lacks an RNA replicase and the host cell constitutively expresses the deleted RNA replicase.

Wherein the host cell is a eukaryotic host cell.

2. The REPLACE system of claim 1, the host cell being an animal host cell.

3. The REPLACE system of claim 1, the host cell being a mammalian host cell.

4. The REPLACE system of claim 1, the host cell being selected from hamster fibroblast cell lines or derived from hamster fibroblast cell lines.

5. The REPLACE system of claim 1, the host cell selected from BHK-21 or derived from BHK-21.

6. The REPLACE system of claim 1, the host cell being a replicase-limited eukaryotic host cell.

7. The REPLACE system of claim 1, the host cell being an animal host cell with limited replicase.

8. The REPLACE system of claim 1, the host cell being a replicase-limited mammalian host cell.

9. The REPLACE system of claim 1, the host cell being selected from a replicase limited hamster fibroblast cell line or derived from a replicase limited hamster fibroblast cell line.

10. The REPLACE system of claim 1, the host cell selected from replicase limited BHK-21 or BHK-21 derived from replicase limited.

11. The REPLACE system of claim 1 further comprising a mutagen that induces a mutation.

12. The REPLACE system of claim 11 wherein the mutagen is selected from any one of a small molecule mutagen, a nucleoside analog.

13. The REPLACE system of claim 11 wherein the mutagen is selected from any one of FAVIPIRAVIR, MOLNUPIRAVIR.

14. A method of RNA replicase-assisted continuous in vivo evolution (REPLACE), comprising:

providing autonomously replicating RNA;

Providing a host cell;

continuously evolving autonomously replicating RNA within the host cell;

Wherein the autonomously replicating RNA is repRNA-v4,

Wherein the autonomously replicating RNA lacks an RNA replicase and the host cell constitutively expresses the deleted RNA replicase,

Wherein the host cell is a eukaryotic host cell.

15. The REPLACE method of claim 14, the host cell being an animal host cell.

16. The REPLACE method of claim 14, the host cell being a mammalian host cell.

17. The REPLACE method of claim 14, the host cell being selected from hamster fibroblast cell lines or derived from hamster fibroblast cell lines.

18. The REPLACE method of claim 14, the host cell being selected from BHK-21 or derived from BHK-21.

19. The REPLACE method of claim 14, the host cell being a replicase-limited eukaryotic host cell.

20. The REPLACE method of claim 14, the host cell being an animal host cell with limited replicase.

21. The REPLACE method of claim 14, the host cell being a replicase-limited mammalian host cell.

22. The REPLACE method of claim 14, the host cell being selected from a replicase limited hamster fibroblast cell line or derived from a replicase limited hamster fibroblast cell line.

23. The REPLACE method of claim 14, the host cell is selected from replicase limited BHK-21 or BHK-21 derived from replicase limited.

24. The REPLACE method of claim 14, further comprising providing a mutagen that induces a mutation.

25. The REPLACE method of claim 24, wherein the mutagen is selected from any one of a small molecule mutagen, a nucleoside analog.

26. The REPLACE method of claim 24, the mutagen being selected from any one of FAVIPIRAVIR, MOLNUPIRAVIR.

27. Use of the REPLACE system of any one of claims 1 to 13 in the manufacture and optimization of biological macromolecules, vaccines, pharmaceuticals, in the generation of a library of mutants, or in the realization of darwinian adaptation.

28. A vector for expressing the autonomously replicating RNA of any one of claims 1-13.

29. A host cell comprising an expression vector for expressing the autonomously replicating RNA of any one of claims 1-13.

30. A vaccine composition comprising RNA produced by or producible by the REPLACE system of any one of claims 1 to 13.

31. A pharmaceutical composition comprising RNA produced by or producible by the REPLACE system of any one of claims 1 to 13.

32. A delivery vector comprising RNA produced by or producible by the REPLACE system of any one of claims 1 to 13.