Progress 07/01/20 to 06/30/23
Outputs
Target Audience:We have presented some of the results generated from this project in various invited seminars and talks. These include: August 12, 2020: Virtual invited seminar; Agricultural Research Organization, Volcani Center, Israel. September 22, 2020: Invited seminar; Innovative Genomics Institute (IGI), UC Berkeley. October 8, 2020: Invited seminar; Seed Central, Center of Seed Excellence and Innovation, UC Davis. November 19, 2020: Virtual invited seminar; Corteva Agriscience. August 2, 2021: Invited talk: Plant Health 2021, American Phytopathological Society, Pittsburgh. December 8, 2021: Virtual invited talk: Crop Bioengineering Center, Iowa State University ISU. December 11, 2021: Virtual plenary talk: XV International Symposium on Virus Diseases of Ornamental Plants. February 3, 2022: Virtual invited seminar: Center for infectious disease dynamics, Penn State University. May 6, 2022: Invited virtual seminar: Center for precision genomics, University of Minnesota. June 5, 2022: Invited talk: Society for in vitro biology (SIVB) meeting, San Diego, CA December 9, 2022: Keynote speaker; Plant Synthetic Biology and Biotechnology, Fort Lauderdale, FL Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project has provided training to a research scientist and postdoctoral fellow in the area of plant virus vector engineering and transgene-free gene editing. The project also provided training to undergraduate students that included the minority students from UC Davis. How have the results been disseminated to communities of interest?Findings from this grant on improved TRV with tRNA-based systems for gene editing have been presented in various invited seminars and talks. We have published two papers and one is in the press. We have another manuscript that will be submitted soon. Over the last three years, we have sent our improved TRV system of gene editing in plants to various researchers in the United States and outside. We have also deposited the vectors developed under this grant to Addgene for distribution to the wider scientific community. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
TRV augmented with mobile RNA sequences induce high efficiency somatic and heritable editing in Nicotiana and Arabidopsis Few months before this grant started, in a collaborative work we showed that the TRV augmented with mobile RNA sequences such as Flowering Locus T (FT) and tRNA-like sequences can induce high efficiency somatic and germline editing in plants expressing Cas9 nuclease (Ellison et al., 2020). Our group specifically reasoned that since some tRNA-like sequences have been shown to be mobile in the phloem and they can transport mRNAs (Zhang et al., 2016), addition of these mobile tRNAs to the single guide RNAs (sgRNAs) could enhance the somatic and heritable editing in plants. Therefore, our group engineered TRV2 with tRNA isoIeucine (tRNAIleu), tRNA methionine (tRNAMet) and tRNA glycine (tRNAGly). Interestingly, we found that inclusion of tRNAIleu and tRNAMet significantly increased the systemic movement of TRV compared to tRNAGly and TRV with no tRNA in Nicotiana benthamiana (Ellison et al., 2020). Furthermore, addition of tRNAIleu to the sgRNAs targeted to phytoene desaturase (PDS) gene showed 80-95% editing efficiency in the somatic tissues. Interestingly, as the TRV with sgRNANbPDS::tRNAIleu moved in the plants, we observed sectors of white photobleaching phenotype because of editing in the PDS gene (Ellison et al., 2020). This phenotype was not observed when TRV with sgRNANbPDS was infected. Moreover, when seeds collected from top 1/3rd of the sgRNANbPDS::tRNAIleu infected plants were set on soil, we observed 80-95% of the seedlings were completely white. These results indicated that sgRNAs augmented with tRNAIleu could induce tetra-allelic editing in a single generation without tissue culture (Ellison et al., 2020). Under this grant, we optimized the TRV-based delivery of sgRNAs for efficient editing in other dicot plants. For efficient introduction of TRV with sgRNAs into Arabidopsis, we optimized a new agro-flooding method that is more efficient than typically used syringe infiltration method. Using this method, our results indicated that sgRNA fused to tRNAIleu moves efficiently into systemic tissue in Arabidopsis compared to sgRNA alone or sgRNA fused to other mobile sequences. TRV with sgRNA targeted to Arabidopsis PDS3 (AtPDS3) fused to tRNAIleu efficiently induced somatic editing. Furthermore, we observed 39-60% heritable biallelic editing in AtPDS3 in the next generation. TRV with sgRNAs targeting Magnesium-chelatase subunit 1 (AtCHLI1) and Magnesium-chelatase subunit 2 (AtCHLI2) involved in chlorophyll synthesis showed efficient somatic editing in both genes indicating feasibility of multiplexing. Since knockout of both AtCHLI1 and AtCHLI2 resulted in lethality, we targeted TRIPTYCHON (AtTRY) and CAPRICE (AtCPC), which function as negative regulators of trichome development and display increased trichomes on leaves of double mutants, leading to a clustered leaf trichomes phenotype. sgRNAs targeting AtTRY and AtCPC resulted in 12-38% biallelic heritable editing phenotype in M1 progenies. Furthermore, 100% of M2 progenies displayed the editing phenotype. These results showed that biallelic mutants can be generated in a single generation and it also facilitates uncovering lethal phenotypes because the phenotype is visible in the original virus infected plants. These findings were published in Plant Physiology (Nagalakshmi et al., 2022). In a collaborative work, we also contributed to show that TRV with tRNAIleu induces efficient heritable base-editing in Arabidopsis. This work was published in Plant Physiology (Liu et al., 2022). TRV augmented with mobile RNA sequences induce high efficiency somatic editing in tomato In order to optimize the editing using the TRV system augmented with tRNA sequences in tomato, we used three different Cas9 expressing tomato lines that were published. Unfortunately, none of these lines produced somatic editing when TRV with sgRNAs targeted to the tomato PHYTOENE DESATURASE (SlPDS3) was delivered to these lines. Our results described above from Nicotiana and Arabidopsis indicated that the level of Cas9 expression is important to obtain good editing when TRV with sgRNA is delivered. In a collaboration with Dr. Staskawicz lab at UC Berkeley, we generated high expressing tomato SpCas9 lines. In this line TRV with sgRNASlPDS::tRNAIleu induces uniform somatic editing. However, our several attempts failed to induce heritable editing in the next generation. We are currently investigating why tomato lines fail to induce heritable editing compared to Nicotiana and Arabidopsis. Engineering TRV1 alone for delivery of gene editing components As part of this grant, we proposed to modify TRV1 to accommodate increased cargo delivery into plants. Since TRV1 alone can infect plants without TRV2, we tested if we could use TRV1 to express sgRNAs for gene editing. For this we tested delivery of sgRNANbPD-tRNAIleu that was cloned just before the 3'UTR of TRV1. We also engineered Pea early browning virus (PEBV) subgenomic promoter into TRV1 to express sgRNAs fused to tRNAIleu. As a control, we cloned sgRNANbPDS without any mobile sequence or subgenomic promoter. Our results show that all of these constructs could induce high efficient somatic and biallelic heritable editing in N. benthamiana and in Arabidopsis. TRV1 is also efficient in multiplex editing of two genes. In addition, TRV1 with sgRNA targeted to NbPDS when combined with TRV2 with sgRNA targeted to AP3, we observed efficient heritable editing in both NbPDS and NbAP3 genes indicating increased number of genes that could be targeted using TRV1 and TRV2 together expressing sgRNAs. The manuscript describing these results is almost ready for submission. We will acknowledge the funding from this grant in the publication. Delivering split Cas nucleases for editing using TRV system Our ultimate goal is to engineer viruses that can deliver both Cas nucleases and sgRNAs for editing in plants. The TRV has a cargo limit of about 2 kb and hence it cannot express 4.2 kb SpCas9. Therefore, we tested a number of split versions of SpCas9 to see if it could induce somatic editing. So far, over 10 combinations we have tested resulted in no somatic editing. We are continuing to explore on how we could get this to express using the TRV system. Delivering smaller Cas nucleases for editing using TRV system To overcome cargo issues with TRV, we have been trying to use small Cas nucleases. Recently several small compact Cas nucleases have been reported. We tested some of these to see if we could deliver these using the TRV system for editing. Even after testing 8-10 guide RNAs targeted to NbPDS, we were not able to see any somatic editing through phenotyping and also by genotyping. However with CasMINI we were able to observe ~10% of somatic editing frequency after testing 10 different sgRNAs targeted to NbPDS. We are currently screening the seeds to evaluate heritable editing efficiency. References Ellison EE, Nagalakshmi U, Gamo ME, Huang P, Dinesh-Kumar SP, Voytas D. (2020) Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nature Plants 6:620-624. Zhang W, Thieme CJ, Kollwig G, Apelt F, Yang L, Winter N, Andersen N, Walther D, Kragler F. (2016) tRNA-related sequences trigger systemic mRNA transport in plants. Plant Cell 28: 1237-1249. Nagalakshmi, U., Meier, N., Liu, J-Y., Voytas, D. F., and Dinesh-Kumar, S. P. (2022) High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus. Plant Physiology 189:1241-1245. Liu, D., Xuan, S., Prichard, L. E., Donahue, L. I., Pan, C., Nagalakshmi, U., Ellison, E. E., Starker, C. G., Dinesh-Kumar, S. P., Qi, Y., and Voytas, D. F. (2022) Heritable base-editing in Arabidopsis using RNA viral vectors. Plant Physiology 189:1920-1924.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Nagalakshmi, U., Meier, N., and Dinesh-Kumar, S. P. (2023) Virus-induced heritable gene editing in plants. Methods Mol. Biol. (in press)
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:We have presented some of the results described in this report in seminars/talks: August 2, 2021: Invited talk: Plant Health 2021, American Phytopathological Society. December 8, 2021: Invited talk: Crop Bioengineering Center, Iowa State University ISU. December 11, 2021: Plenary talk: XV International Symposium on Virus Diseases of Ornamental Plants. February 3, 2022: Seminar: Center for infectious disease dynamics, Penn State University. May 6, 2022: Seminar: Center for precision genomics, University of Minnesota. June 5, 2022: Invited talk: Society for in vitro biology (SIVB) meeting. Changes/Problems:Full and partial campus closer as part of mitigating COVID-19 in the last two years impacted this grant. What opportunities for training and professional development has the project provided?Project has provided training to a research scientist and a postdoctoral fellow in the area of plant virus genome engineering and gene editing. The project also provided training to three undergraduate students from UC Davis. How have the results been disseminated to communities of interest?Our findings have been presented in various seminars and invited talks at the conference. During the reporting period, two manuscripts have been published. What do you plan to do during the next reporting period to accomplish the goals?We will continue to optimize methods to deliver sgRNAs for heritable editing in tomato using TRV2. We will continue our efforts on engineering of TRV1 to deliver sgRNAs and possibly part of Cas nuclease. We will continue to test if we can deliver smaller Cas nucleases through the TRV system for transgene free editing in plants. In addition, we will test delivery of split Cas nucleases for editing. We are also re-engineering Sonchus yellow net rhabdovirus (SYNV) to express both Cas nuclease and sgRNAs for heritable editing in N. benthamiana plants.
Impacts
What was accomplished under these goals?
High efficiency biallelic heritable gene editing and base editing using TRV system In the last report, we reported that Tobacco rattle virus (TRV) augmented with mobile RNA sequences such as Flowering Locus T (FT) and tRNA-like sequences can induce high efficiency somatic and germline editing in Nicotiana benthamiana plants expressing SpCas9 nuclease (Ellison et al., 2020). During this review period, we have optimized the TRV-based delivery of single guide RNAs (sgRNAs) for efficient editing in Arabidopsis plants. For efficient introduction of TRV with sgRNAs into Arabidopsis, we optimized a new agro-flooding method that is more efficient than typically used syringe infiltration method. Using this method, our results indicated that sgRNA fused to tRNA isoleucine (tRNAIleu) moves efficiently into systemic tissue in Arabidopsis compared to sgRNA alone or sgRNA fused to other mobile sequences. TRV with sgRNA targeted to PHYTOENE DESATURASE3 (AtPDS3) fused to tRNAIleu efficiently induced somatic editing. Furthermore, we observed 39-60% heritable biallelic editing in AtPDS3 in the next generation. TRV with sgRNAs targeting Magnesium-chelatase subunit 1 (AtCHLI1) and Magnesium-chelatase subunit 2 (AtCHLI2) involved in chlorophyll synthesis showed efficient somatic editing in both genes indicating feasibility of multiplexing. Since knockout of both AtCHLI1 and AtCHLI2 resulted in lethality, we targeted TRIPTYCHON (AtTRY) and CAPRICE (AtCPC), which function as negative regulators of trichome development and display increased trichomes on leaves of double mutants, leading to a clustered leaf trichomes phenotype. sgRNAs targeting AtTRY and AtCPC resulted in 12-38% biallelic heritable editing phenotype in M1 progenies. Furthermore, 100% of M2 progenies displayed the editing phenotype. The optimized and simple agro-flooding method combined with TRV augmented with tRNAIleu is very efficient for inducing somatic and heritable multiplex editing in SpCas9 expressing Arabidopsis. Biallelic mutants can be generated in a single generation and it also facilitates uncovering lethal phenotypes because the phenotype is visible in the original virus infected plants. Although TRV can transiently invade the meristem, the addition of tRNAIleu is essential to achieve efficient heritable editing in N. benthamiana and in Arabidopsis. The results described above was recently published (Nagalakshmi et al., 2022). In a collaborative work, we also contributed to show that TRV with tRNAIleu induces efficient heritable base-editing in Arabidopsis. This work was recently published (Liu et al., 2022). Currently, we are optimizing the TRV system to deliver sgRNAs into Cas9 expressing tomato lines that was developed by Dr. Staskawicz's group at UC Berkeley. Engineering TRV1 alone for delivery of gene editing components As part of this grant, we proposed to modify TRV1 to accommodate increased cargo delivery into plants. Since TRV1 alone can infect plants without TRV2, we tested if we could use TRV1 to express sgRNAs for gene editing. For this we tested delivery of sgRNA targeted to NbPDS fused to tRNAIleu (sgRNANbPD-tRNAIleu) that was cloned just before the 3'UTR of TRV1. We also engineered Pea early browning virus (PEBV) subgenomic promoter into TRV1 to express sgRNAs fused to tRNAIleu. As a control, we cloned sgRNANbPDS without any mobile sequence or subgenomic promoter. Our results show that both sgRNANbPD-tRNAIleu and PEBV expressed sgRNANbPD-tRNAIleu could induce high efficient somatic and biallelic heritable editing in N. benthamiana and in Arabidopsis. TRV1 is also efficient in multiplex editing of two genes. In addition, TRV1 with sgRNA targeted to NbPDS when combined with TRV2 with sgRNA targeted to AP3, we observed efficient heritable editing in both NbPDS and NbAP3 genes indicating increased number of genes that could be targeted using TRV1 and TRV2 together expressing sgRNAs. We are currently preparing a manuscript describing results for publication. Currently, we are engineering TRV1 and TRV2 to see if we could engineer split Cas9 fragments to express Cas9 protein along with sgRNAs to induce editing. Delivering smaller Cas nucleases for editing using TRV system During the last year, several small Cas nucleases have been reported. We are actively testing these nucleases using TRV to see if we can express these Cas proteins for efficient editing using N. benthamiana system. References Ellison EE, Nagalakshmi U, Gamo ME, Huang P, Dinesh-Kumar SP, Voytas D. (2020) Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nature Plants 6:620-624. Nagalakshmi, U., Meier, N., Liu, J-Y., Voytas, D. F., and Dinesh-Kumar, S. P. (2022) High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus. Plant Physiology Apr 7:kiac159. doi: 10.1093/plphys/kiac159. Online ahead of print. Liu, D., Xuan, S., Prichard, L. E., Donahue, L. I., Pan, C., Nagalakshmi, U., Ellison, E. E., Starker, C. G., Dinesh-Kumar, S. P., Qi, Y., and Voytas, D. F. (2022) Heritable base-editing in Arabidopsis using RNA viral vectors. Plant Physiology May 5:kiac206. doi: 10.1093/plphys/kiac206. Online ahead of print.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Nagalakshmi, U., Meier, N., Liu, J-Y., Voytas, D. F., and Dinesh-Kumar, S. P. (2022) High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus. Plant Physiology Apr 7:kiac159. doi: 10.1093/plphys/kiac159. Online ahead of print.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Liu, D., Xuan, S., Prichard, L. E., Donahue, L. I., Pan, C., Nagalakshmi, U., Ellison, E. E., Starker, C. G., Dinesh-Kumar, S. P., Qi, Y., and Voytas, D. F. (2022) Heritable base-editing in Arabidopsis using RNA viral vectors. Plant Physiology May 5:kiac206. doi: 10.1093/plphys/kiac206. Online ahead of print.
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Progress 07/01/20 to 06/30/21
Outputs
Target Audience:We have presented some of the results described in this report in seminars/talks: August 12, 2020: Agricultural Research Organization - the Volcani Center, Israel September 22, 2020: Innovative Genomics Institute, UC Berkeley October 8, 2020: Seed Central, Center of Seed Excellence and Innovation November 19, 2020: Corteva Agriscience Changes/Problems:Immediately after the grant was recommended for funding, UC Davis at the end of March 2020 completely closed down the research activities because of COVID-19 pandemic. Only one person from the lab was allowed to water plants and maintain important plant stocks but not any research work. On June 1, 2020, the UC Davis allowed 33% of the personnel to work in the lab at any given time; this means each person could work about 3-4 hours per day because of different ongoing projects in the laboratory. On March 25, 2021 the occupancy was increased to 66%. Only recently on June 15, the UC Davis campus allowed research operations to the pre-covid level. The above described shut downs and limited occupancy in the laboratory have significantly affected the progress of this grant. In addition, COVID restrictions affected recruitment proecess of post-doc to work under this project. Despite these issues we have made significant progress in this grant. What opportunities for training and professional development has the project provided?Project has provided training to a research scientist and a postdoctoral fellow in the area of plant virus genome engineering and gene editing. The project also provided training to a Hispanic background undergraduate student from UC Davis. How have the results been disseminated to communities of interest?tRNA-based augmentation of sgRNA delivery through TRV has been presented in various seminars and talks. Currently, we are preparing a manuscript describing the results of Arabidopsis. What do you plan to do during the next reporting period to accomplish the goals?We will continue to optimize methods to deliver sgRNAs for heritable editing in tomato using TRV2. We will continue our efforts on engineering of TRV1 to deliver sgRNAs and possibly part of Cas nuclease. We will continue to test if we can deliver smaller Cas nucleases such as Cas-phi and Cas-12f1 through the TRV system for transgene free editing in plants. In addition, we will test delivery of split Cas nucleases for editing. Last year Zhenghe Li's group reported that Sonchus yellow net rhabdovirus (SYNV) could be used to deliver both Cas9 nuclease and sgRNA into N. benthamiana plants (Ma et al., 2020, Nature Plants 6: 773-779). However, this virus system cannot result in heritable editing. To generate edited plants, the tissue from SYNV infected plants should be regenerated through a tissue culture-based approach. This work showed that 90% of the regenerated plants contained targeted mutations. In the coming months, we will engineer SYNV vectors from Zhenghe Li's group to see if we can modify the SYNV system using our mobile tRNA sequences so that it can induce heritable editing without going through the tissue culture step.
Impacts
What was accomplished under these goals?
Most of the data presented here is unpublished and hence should not be made public. TRV augmented with mobile RNA sequences induce high efficiency somatic and heritable editing in plants Just before this grant was awarded, in a collaborative work we showed that the TRV vector augmented with mobile RNA sequences such as Flowering Locus T (FT) and tRNA-like sequences can induce high efficiency somatic and germline editing in plants expressing Cas9 nuclease (Ellison et al., 2020). Our group specifically reasoned that since some tRNA-like sequences have been shown to be mobile in the phloem and they can transport mRNAs (Zhang et al., 2016), addition of these mobile tRNAs to the single guide RNAs (sgRNAs) could enhance the somatic and heritable editing in plants. Therefore, our group engineered TRV2 with tRNA Ieucine (tRNAIleu), tRNA methionine (tRNAMet) and tRNA glycine (tRNAGly) to determine if addition of these sequences at the end of sgRNAs could enhance somatic and heritable gene editing. Interestingly, we found that inclusion of tRNAIleu and tRNAMet significantly increased the systemic movement of TRV compared to tRNAGly and TRV with no tRNA in Nicotiana benthamiana (Ellison et al., 2020). Furthermore, addition of tRNAIleu to the sgRNAs targeted to phytoene desaturase (PDS) gene showed 80-95% editing efficiency in the somatic tissues of TRV infected plants. Since N. benthamiana contains two similar copies of PDS genes, the editing should be achieved in both genes to impart white photobleaching phenotype. Interestingly, as the TRV with sgRNANbPDS::tRNAIleu moved in the plants, we observed sectors of white photobleaching phenotype because of editing in the PDS gene (Ellison et al., 2020). This phenotype was not observed when TRV with sgRNANbPDS was infected. Moreover, when seeds collected from top 1/3rd of the sgRNANbPDS::tRNAIleu infected plants were set on soil, we observed 80-95% of the seedlings were completely white. These results indicated that sgRNAs augmented with tRNAIleu could induce tetra-allelic editing in a single generation without tissue culture (Ellison et al., 2020). Under this grant, we have been optimizing the TRV augmented with tRNA sequences to induce high efficiency editing in other plants including tomato. In Arabidopsis, we have previously shown that infiltration of 2-3 leaf stage plants with TRV can efficiently induce silencing of genes based on virus-induced gene silencing (VIGS) approach (Burch-Smith et al., 2006). Although the method used in this paper to deliver TRV with sgRNAs augmented with tRNAs works, the efficiency of infection is very low. Therefore, we optimized methods for efficient introduction of TRV to Arabidopsis. As opposed to the N. benthamiana system, FT mobile RNA augmented TRV does not enhance TRV systemic movement but tRNAIleu and tRNAMet significantly enhances systemic TRV movement. This is consistent with the published work that FT protein moves but not FT RNA in Arabidopsis. Using the optimized method we can efficiently induce high frequency of heritable editing in single, double and triple genes in Arabidopsis. We are currently preparing a manuscript describing these results for submission. Currently, we have three different Cas9 expressing tomato lines that we are using to optimize the editing using a TRV system augmented with tRNA sequences. The results from pilot experiments indicate that the methods we have used in N. benthamiana and Arabidopsis may not be efficient in tomatoes. Therefore, we are currently trying different methods to optimize the TRV-based sgRNA delivery into tomatoes. Engineering TRV1 alone for delivery of gene editing components As part of this grant, we proposed to modify TRV1 to accommodate increased cargo delivery into plants. First, we optimized and tested if we could use TRV1 alone without TRV2 to deliver sgRNAs into plants for editing. For this we engineered Pea early browning virus (PEBV) subgenomic promoter that we have used in the TRV2 to deliver sgRNAs just after 16K open reading frame (ORF) stop codon. We cloned sgRNANbPDS or sgRNANbPDS::tRNAIleu into TRV1 after 16K with and without pPEBV promoter. Surprisingly, sgRNAPDS alone without tRNAIleu delivered through TRV1 can induce high efficiency somatic editing in N. benthamiana plants. The efficiency is same as delivering sgRNANbPDS with tRNAIleu and under the control of pPEBV promoter. Currently, we are waiting to collect seeds to evaluate the heritability of editing efficiency. In addition, we are investigating the mechanistic basis of how TRV1 can deliver sgRNAs into growing parts of the plant compared to sgRNA delivered through TRV2. Outcome of these experiments should inform us on how to engineer TRV1 to increase cargo capacity. In another approach to increase the cargo capacity of TRV1, we have generated deletions within TRV1 to generate smaller amplicons. Our initial findings indicate that we can delete the majority of TRV1 and when we deliver this deletion expressing sgRNANbPDS with TRV1+TRV2, it can induce somatic editing. Currently, we are estimating heritability and also testing this vector to see if we can deliver larger cargoes such as Cas nucleases. Delivering smaller Cas nucleases for editing using TRV system In July of 2020, Doudna's group reported a compact Cas nuclease called Cas-phi from large phage genomes (Pausch et al., 2020). Considering Cas-phi is about 70 kDa (half the size of SpCas9), we are testing if we could deliver this Cas for editing using the TRV system. We have generated TRV2 with Cas-phi nuclease and 8 different sgRNAs targeted to PDS. We are currently testing if any of these sgRNAs when coexpressed with Cas-phi induces somatic editing in the infiltrated leaves. If we do see positive editing, we will test those sgRNAs for systemic somatic editing and heritability. In addition to Cas-phi we are also testing another smaller Cas nuclease called Cas12f1 (also known as Cas14a) that has been recently shown to cleave double stranded DNA targets (Takeda et al., 2021). References Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP. (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol. 142: 21-27. Ellison EE, Nagalakshmi U, Gamo ME, Huang P, Dinesh-Kumar SP, Voytas D. (2020) Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nature Plants 6:620-624. Pausch P, Al-Shayeb B, Bisom-Rapp E, Tsuchida CA, Li Z, Cress BF, Knott GJ, Jacobsen SE, Banfield JF, Doudna JA. (2020) CRISPR-Cas-phi from huge phages is a hypercompact genome editor. Science 369: 333-337. Zhang W, Thieme CJ, Kollwig G, Apelt F, Yang L, Winter N, Andersen N, Walther D, Kragler F. (2016) tRNA-related sequences trigger systemic mRNA transport in plants. Plant Cell 28: 1237-1249. Takeda SN, Nakagawa R, Okazaki S, Hirano H, Kobayashi K, Kusakizako T, Nishizawa T, Yamashita K, Nishimasu H, Nureki O. (2021) Structure of the miniature type V-F CRISPR-Cas effector enzyme. Mol Cell 81: 558-570.
Publications
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