Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs

Abstract

An in planta gene editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are augmented with sequences that promote cell-to-cell mobility. Mutant progeny are recovered in the next generation at frequencies ranging from 65 to 100%; up to 30% of progeny derived from plants infected with a virus expressing three sgRNAs have mutations in all three targeted loci.

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Fig. 1: Heritable gene editing using RNA viruses and mobile sgRNAs.
Fig. 2: Multiplexed heritable gene editing using an RNA virus and mobile sgRNAs.

Data availability

High-throughput sequencing data have been deposited in the NCBI Sequence Read Archive database under BioProject accession no. PRJNA625335. TRV vectors are available from Addgene.

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Acknowledgements

This work was supported by grant no. HR0011-17-2-0053 from the Defense Advanced Research Projects Agency (DARPA; to D.F.V. and S.D.-K.), funds from the Innovative Genomics Institute grant (to S.D.-K.) and National Science Foundation grant no. NSF-IOS-1339185 (to S.D.-K.). The University of Minnesota and University of California, Davis are part of a team supporting DARPA’s Insect Allies Program under agreement no. HR0011-17-2-0053. We thank J.-Y. Liu for maintaining plants and for technical help during the revision of the manuscript. We thank M. Leffler for help with the figures.

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Authors

Contributions

E.E.E. designed the research, performed experiments with assistance from M.E.G., analysed data and wrote the manuscript. The work with tRNAs was carried out by U.N. and P.-J.H. D.F.V. and S.D.-K. supervised the research and helped write the manuscript.

Corresponding author

Correspondence to Daniel F. Voytas.

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Competing interests

E.E.E. and D.F.V. are named inventors on a patent application pertaining to technology filed by the University of Minnesota. D.F.V. serves as Chief Science Officer for Calyxt, an agricultural biotechnology company that uses gene editing to create new crop varieties.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Addition of Flowering Locus T sequences to sgRNAs does not inhibit editing activity.

There are no significant differences in editing efficiency when the sgRNA is either unmodified (n = 5 independent biological replicates, \(\bar x\) = 0.73 ± 0.27) or modified with FT (n = 3, \(\bar x\) = 0.61 ± 0.19, p-value= 0.477), mFT (n = 5, \(\bar x\) = 0.87 ± 0.05, p-value= 0.328), or truncated FT (n = 3, \(\bar x\) = 0.88 ± 0.03, p-value= 0.286). The modified or unmodified sgRNAs were expressed from TRV vectors, and tissue was collected from the infiltrated site in N. benthamiana leaves. Collecting tissue from the infiltrated site allows for examination of sgRNA function alone. DNA was extracted from the infiltrated site two weeks after infiltration. The targeted locus was PCR amplified and sequenced using Next Generation Sequencing (NGS). The Y axis denotes the fraction, displayed as a percentage, of reads with indels at the targeted locus divided by the total number of reads. Each dot represents one plant replicate. Bars indicate the mean editing frequency; error bars represent ± standard deviation. Asterisks indicate statistically significant differences compared with the unmodified sgRNA by the two-sided Student’s t-test, NS > 0.05, *<0.05, **<0.01.

Extended Data Fig. 2 Modified sgRNAs enable high frequencies of somatic and heritable editing at the AG locus.

a, Editing efficiency of vectors targeting AG at the eighth systemic leaf. sgRNAs modified with FT (n = 3 independent biological replicates, \(\bar x\) = 0.73 ± 0.19), mFT (n = 3, \(\bar x\) = 0.81 ± 0.09), or Truncated FT (n = 2, \(\bar x\) = 0.85 ± 0.03) yield higher average editing frequencies and lower variance compared to unmodified sgRNAs (n = 4, \(\bar x\) = 0.29 ± 0.29). Tissue was collected approximately 6 weeks after infiltration. The Y axis is the percentage of NGS reads with indels divided by the total number of reads. Each dot represents one plant replicate. Bars indicate the mean editing frequency; error bars represent ± standard deviation. b, sgRNAs modified with FT (n = 3 independent biological replicates, \(\bar x\) = 0.89 ± 0.10, p-value= 0.002) or mFT (n = 3, \(\bar x\) = 0.87 ± 0.22, p-value= 0.011) or Truncated FT (n = 2, \(\bar x\) = 0.81 ± 0.06, p-value= 0.004) produce significantly higher frequencies of heritable editing compared to unmodified sgRNAs (n = 3, \(\bar x\) = 0.10 ± 0.13). In some cases, 100% of progeny contained a mutation. Heritable editing frequency is the fraction, displayed as a percentage, of progeny that contained an indel in at least one allele divided by the total number of progeny genotyped. Each dot represents the heritable editing frequency in progeny of one parent replicate infected with TRV expressing either unmodified sgRNAs (n = 13, 16, or 17) or modified with FT (n = 20, 11, or 14 progeny), mFT (n = 18, 21, or 19) or Truncated FT (n = 20 or 17). Bars indicate the mean editing frequency; error bars represent ± standard deviation. Asterisks indicate statistically significant differences compared to the unmodified sgRNA by the two-sided Student’s t-test, *<0.05, **<0.01. (c) Modified sgRNAs produce a higher portion of bi-allelic and heterozygous mutations compared to unmodified sgRNAs. In one case, 100% of progeny contained bi-allelic mutations. The percentage of each zygosity was determined by the fraction of progeny that contained that genotype divided by total progeny assessed. The same progeny used to determine heritable editing frequency were used to calculate the genotype percentages.

Extended Data Fig. 3 Inheritance of gene edits at the N. benthamiana AG locus.

a, Genotypes of progeny from parents with observed +T/-7bp mutations. One seedling (M1) produced from a parent plant (M0) infected with TRV vectors expressing a sgRNA with Truncated FT had +T/-7bp mutations in AG. This plant was grown to maturity and seeds were collected from the first seed pod. Nineteen progeny of this plant were genotyped, and an approximate Mendelian inheritance was observed. b, Genotypes of progeny from parents with observed +T/ + T mutations. One seedling (M1) from a parent plant (M0) infected with TRV vectors expressing a sgRNA with truncated FT had +T/ + T mutations in AG alleles. This plant was grown to maturity and seeds were collected from the first seed pod. Nineteen progeny of this plant were genotyped, and the expected Mendelian inheritance was observed. Genotypes were determined through DNA extraction of each progeny, amplification of the AG locus, Sanger sequencing, and detection of indels by ICE (Synthego). Due to noise in ICE analyses, the following parameters were used to call genotype: 0%-10%=WT, 35%-65%= heterozygous (WT/indel), 85%-100%=bi-allelic mutation.

Extended Data Fig. 4 Phenotypes of plants infected with TRV vectors expressing sgRNAs that target N. benthamiana PDS.

Pictures of plants were taken between four and eight weeks after infection. The pictures display some of the typical phenotypes observed from either a, a non-infected plant or plants infected with a TRV vector expressing b, a non-augmented sgRNA, c, an FT-augmented sgRNA, d, a mFT-augmented sgRNA, e, a truncated FT-augmented sgRNA, f, a glycine tRNA-augmented sgRNA, g, a methionine tRNA-augmented sgRNA, h, an isoleucine tRNA-augmented sgRNA. Similar phenotypes were observed in three independent experiments, and each experiment consisted of 2–3 independent plants.

Extended Data Fig. 5 TRV2 abundance reflects heritable editing efficiency.

a, Relative TRV2 RNA levels were measured by quantitative real-time PCR relative to the control, PP2A. b, Green and bleached plant phenotypes of progeny from parental plants infected with a TRV2 vector expressing sgNbPDS, sgNbPDS-tRNAIleu, sgNbPDS-tRNAMet, or sgNbPDS-tRNAGly. Frequencies of heritable editing correlate with viral abundance in the parent generation. About 50–60 progeny seeds from a pod from the top of the parent plants were planted on soil in individual pots. Pictures were taken 10–14 days post-seeding. This experiment was repeated twice with similar results.

Extended Data Fig. 6 Mutation types identified at the NbPDS locus in progeny plants derived from a parental plant infected with sgNbPDS-tRNAIleu or with sgNbPDS-tRNAMet.

DNA fragments flanking the NbPDS target site from progeny plants were cloned and 6–10 clones were sequenced. For each plant, the wild type sequence is shown at the top and the target site is shown in red and the protospacer adjacent motif (PAM) is underlined. Number of base addition (+) or deletion (−) are shown on the right.

Extended Data Fig. 7 TRV is not transmitted to the next generation.

RT-PCR was performed using total RNA extracted from progeny heterozygous for mutations at PDS of plants infected with TRV editing vectors. a, Progeny RNA from a parental plant infected with TRV-sgNbPDS-tRNAMet (lanes 1–2) and TRV-sgNbPDS-tRNAIleu (lanes 3–4), TRV-infected control (lane 5) and mock-infected control plant tissue (lane 6) using primers that amplify the TRV 3’-end (upper panel) and endogenous NbPP2A gene (bottom panel). Lane 7, TRV plasmid template used in PCR as a positive control. b, Progeny RNA from parental plant replicate 1 infected with TRV-sgNbPDS-FT (lanes 10–17), TRV-infected control (lanes 9, 19, and 29) and water control (lanes 8, 18, and 28) using primers that amplify TRV across the sgNbPDS-FT insert (upper panel) and endogenous NbEF1a gene (bottom panel). c, Progeny RNA from parental plant replicate 2 infected with TRV-sgNbPDS-FT (lanes 20–27). d, Progeny RNA from parental plant replicate 3 infected with TRV-sgNbPDS-FT (lanes 30–37). Lanes M1, 100 bp DNA ladder; M2, 1 kb DNA ladder; M3, NEB 1 kb+ ladder. Conclusions were the same for experiments performed at different institutions: the experiment in panel a was performed at UC Davis; the experiments in panels b–d were performed at the University of Minnesota.

Extended Data Fig. 8 Heritable editing frequency increases as the infected plant matures.

a, Green and bleached plant phenotypes of progeny from parental plants infected with a TRV vector expressing sgNbPDS-tRNAIleu, sgNbPDS-tRNAMet, or sgNbPDS-tRNAGly. About 50–60 progeny seeds from a pod from the top of the parent plants (top panels), a pod from middle of the parent plants (middle panels) and a pod from base of the parent plants (bottom panels) were planted on soil in individual pots. Pictures were taken 10–14 days post-seeding. Similar phenotypes were observed in two independent experiments. b, Ratio of bleached progeny to total number of progeny from individual seed pods. Parental plants were infected with a TRV vector expressing sgNbPDS (Unmodified sgRNA, n = 3 independent biological replicates),sgNbPDS-Truncated FT (TRV2-NbPDS-AtFT, n = 3), sgNbPDS-tRNAIleu (n = 2), sgNbPDS-tRNAMet (n = 2), or sgNbPDS-tRNAGly (n = 2). Seeds were collected from each seed pod individually with Seed Pod 1 being the first emerged seed pod and ascending vertically. Thirty to sixty progeny were phenotyped per pod. Three infected parent replicates were collected per treatment. Each shape corresponds to one infected parent replicate. Each line corresponds to the mean percentage of bleached progeny from each modified or unmodified sgRNA treatment.

Extended Data Fig. 9 Growth conditions affect heritable editing frequency.

Bleached progeny (%) indicates the ratio of bleached progeny to green progeny. Parental plants were infected with a TRV vector expressing sgNbPDS (TRV2-sgNbPDS) or, sgNbPDS-Truncated FT (TRV2-sgNbPDS-AtFT). Progeny were collected from the entire infected parent plant and 150–300 seedlings were phenotyped. TRV2-sgNbPDS infected plants were grown at two different conditions: 22 °C with 24 h light (n = 2 independent biological replicates, \(\bar x\) = 0.004 ± 0.005) or 26 °C day/22 °C night with a 12 hour day/night cycle (n = 3, \(\bar x\) = 0.034 ± 0.039). All TRV2-sgNbPDS-AtFT were grown at 26 °C day/22 °C night with a 12 hour day/night cycle (n = 3, \(\bar x\) = 0.421 ± 0.125). Each dot represents one plant replicate. Bars indicate the mean editing frequency; error bars represent ± standard deviation.

Extended Data Fig. 10 Method for rapid cloning of augmented sgRNAs into TRV vectors.

A cloning vector was developed that allows for simple and efficient cloning of unique sgRNA target sequences into TRV2 vectors. Unique sgRNAs are added to the vector using one unique primer (that contains the sgRNA target of interest) and one reverse primer (that can be used for all unique primers). These primers are used to amplify the modified sgRNA from the TRV2 cloning vector backbone. This amplicon is then added to the TRV2 cloning vector through a Golden Gate Assembly cloning step to create a final TRV2 vector with the modified sgRNA of interest.

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Ellison, E.E., Nagalakshmi, U., Gamo, M.E. et al. Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nat. Plants 6, 620–624 (2020). https://doi.org/10.1038/s41477-020-0670-y

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