Frequently Asked Questions
Questions
Where can I download the cloning methods?
Where do I order B/c vectors compatible with P-SAMS oligonucleotides?
Are P-SAMS oligonucleotides compatible with other amiRNA cloning vectors?
What B/c vectors are compatible with my species?
Should I design an artificial miRNA or several synthetic tasiRNAs?
What is the difference between Optimal results and Suboptimal results?
How many synthetic tasiRNA can I multiplex into one construct?
Answers
Q: How does P-SAMS work?
A: The P-SAMS process is virtually the same whether designing an artificial microRNA (amiRNA) or a synthetic trans-acting short-interfering RNA (syn-tasiRNA) to target one or more genes of interest. P-SAMS first identifies all possible target sites by cataloging the complete set of 21-nucleotide sequences from all input transcripts, including isoforms (foreground set). If off-target filtering is enabled, the foreground target site set is filtered to remove sites that contain a 15-nucleotide sequence from positions 6–20 (core target pairing sequence) that perfectly match a transcript that is not contained in the input set (background set). The remaining sites are grouped by the core target pairing sequence, and only site groups that contain all input genes are considered further. Grouped sites are scored and ranked based on group-wise similarity and the identify of nucleotides at specific positions (positions 1, 2, 3 and 21). For each group of sites, a guide RNA (amiRNA or syn-tasiRNA) is designed to target all sites with the additional criteria that i) the guide RNA has a 5’U nucleotide, ii) position 19 of the guide is a C (so that the guide RNA* has a 5’G), and iii) position 21 is intentionally mismatched. Finally, P-SAMS uses TargetFinder to predict target RNAs for each guide, and guide RNAs that are only predicted to target transcripts from input genes are output as optimal results. Currently only up to three optimal results are returned due to the relatively long runtime requirements of TargetFinder.
Q: How do I cite P-SAMS?
A: If using P-SAMS please cite:
Fahlgren, N, Hill ST, Carrington JC, Carbonell A (2016) P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design. Bioinformatics 32: 157-158. doi: 10.1093/bioinformatics/btv534.
If using AtMIR390a- or AtTAS1c-based B/c vectors please cite:
Carbonell A, Takeda A, Fahlgren N, Johnson SC, Cuperus JT, Carrington JC (2014) New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiology 165: 15–29. doi: 10.1104/pp.113.234989.
If using OsMIR390-based B/c vectors please cite:
Carbonell A., Fahlgren N, Mitchell S, Cox Jr KL, Reilly KC, Mockler TC, Carrington JC (2015) Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric MIRNA precursors. The Plant Journal 82: 1061-1075. doi: 10.1111/tpj.12835.
Q: Where can I download the cloning methods?
A: The cloning protocol is available here.
Q: What are B/c vectors?
A: BsaI-ccdB ('B/c') vectors are used for direct cloning of artificial microRNA (amiRNA) or synthetic trans-acting short-interfering RNA (syn-tasiRNA). The majority of B/c vectors are plant expression vectors with a unique combination of promoter and terminator sequences for expressing amiRNA or syn-tasiRNA, and bacterial and plant antibiotic resistance genes. We also developed a subset of GATEWAY-compatible entry B/c vectors to clone the amiRNA or syn-tasiRNA insert and subsequently recombine it into the preferred GATEWAY expression vector containing a promoter, terminator or other features of choice. B/c vectors contain a modified version of an amiRNA or syn-tasiRNA precursor sequence that includes a ccdB cassette flanked by two BsaI sites. AmiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background B/c vector. P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer output the sequence of the two oligonucleotides needed to generate the amiRNA or syn-tasiRNA insert, respectively. B/c amiRNA vectors for eudicots and monocots contain the Arabidopsis thaliana MIR390a or Oryza sativa MIR390 precursor sequence, respectively. B/c syn-tasiRNA vectors contain the Arabidopsis thaliana TAS1c precursor sequence.
Q: Where do I order B/c vectors compatible with P-SAMS oligonucleotides?
A: The following amiRNA and syn-tasiRNA B/c vectors are available from Addgene: pENTR-AtMIR390a-B/c (Addgene plasmid 51778), pMDC32B-AtMIR390a-B/c (Addgene plasmid 51776), pMDC123SB-AtMIR390a-B/c (Addgene plasmid 51775), pFK210B-AtMIR390a-B/c (Addgene plasmid 51777), pENTR-AtTAS1c-B/c (Addgene plasmid 51774), pMDC32B-AtTAS1c-B/c (Addgene plasmid 51773), pMDC123SB-AtTAS1c-B/c (Addgene plasmid 51772), pENTR-OsMIR390-B/c (Addgene plasmid 61468), pMDC32B-OsMIR390-B/c (Addgene plasmid 61467), pMDC123SB-OsMIR390-B/c (Addgene plasmid 61466), and pH7WG2B-OsMIR390-B/c (Addgene plasmid 61465).
Q: Are P-SAMS oligonucleotides compatible with other amiRNA cloning vectors?
A: No. P-SAMS oligonucleotides are only compatible with B/c vectors. If you want to clone your artificial miRNA or synthetic tasiRNA into a different vector system, you can still use P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer apps to design the artificial miRNA or synthetic tasiRNA sequences, respectively.
Q: What B/c vectors are compatible with my species?
A: Check the following tables to determine the species compatibility of each B/c vector.
| Eudicot amiRNA vectors: BsaI/ccdB-based ('B/c') vectors for direct cloning of amiRNAs to use in eudicot species. CaMV, Cauliflower mosaic virus; nos, nopaline synthase; rbcS, Rubisco small subunit. |
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|---|---|---|---|---|---|---|---|---|
| Vector | Bacterial antibiotic resistance | Plant antibiotic resistance | GATEWAY use | Promoter | Terminator | Plant species tested | Addgene number and link | Plasmid sequence |
| pENTR-AtMIR390a-B/c | Kanamycin | - | Donor | - | - | - | 51778 | FASTA GenBank |
| pFK210B-AtMIR390a-B/c | Spectinomycin | BASTA | - | CaMV 35S | rbcS | A. thaliana | 51777 | FASTA GenBank |
| pMDC123SB-AtMIR390a-B/c | Kanamycin | BASTA | - | CaMV 2x35S | nos | A. thaliana N. benthamiana |
51775 | FASTA GenBank |
| pMDC32B-AtMIR390a-B/c | Kanamycin Hygromycin |
Hygromycin | - | CaMV 2x35S | nos | A. thaliana N. benthamiana |
51776 | FASTA GenBank |
| Monocot amiRNA vectors: OsMIR390-BsaI/ccdB ('B/c') vectors for direct cloning of amiRNAs to use in monocot species. CaMV, Cauliflower mosaic virus; nos, nopaline synthase; Os, Oryza sativa. |
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|---|---|---|---|---|---|---|---|---|
| Vector | Bacterial antibiotic resistance | Plant antibiotic resistance | GATEWAY use | Promoter | Terminator | Plant species tested | Addgene number and link | Plasmid sequence |
| pENTR-OsMIR390-B/c | Kanamycin | - | Donor | - | - | - | 61468 | FASTA GenBank |
| pMDC123SB-OsMIR390-B/c | Kanamycin | BASTA | - | CaMV 2x35S | nos | N. benthamiana | 61466 | FASTA GenBank |
| pMDC32B-OsMIR390-B/c | Kanamycin Hygromycin |
Hygromycin | - | CaMV 2x35S | nos | N. benthamiana B. distachyon |
61467 | FASTA GenBank |
| pH7WG2B-OsMIR390-B/c | Spectinomycin | Hygromycin | - | Os Ubiquitin | CaMV | B. distachyon | 61465 | FASTA GenBank |
| syn-tasiRNA vectors: OsMIR390-BsaI/ccdB ('B/c') vectors for direct cloning of syn-tasiRNAs to use in Arabidopsis thaliana and closely related species*. CaMV, Cauliflower mosaic virus; nos, nopaline synthase. |
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|---|---|---|---|---|---|---|---|---|
| Vector | Bacterial antibiotic resistance | Plant antibiotic resistance | GATEWAY use | Promoter | Terminator | Plant species tested | Addgene number and link | Plasmid sequence |
| pENTR-AtTAS1c-B/c | Kanamycin | - | Donor | - | - | - | 51774 | FASTA GenBank |
| pMDC123SB-AtTAS1c-B/c | Kanamycin | BASTA | - | CaMV 2x35S | nos | A. thaliana N. benthamiana* |
51772 | FASTA GenBank |
| pMDC32B-AtTAS1c-B/c | Kanamycin Hygromycin |
Hygromycin | - | CaMV 2x35S | nos | A. thaliana N. benthamiana* |
51773 | FASTA GenBank |
| *miR173-guided cleavage is required to trigger tasiRNA biogenesis but is only conserved in species closely related to A. thaliana. A construct expressing miR173 has to be co-expressed with the syn-tasiRNA construct to trigger syn-tasiRNA biogenesis in species that lack miR173. | ||||||||
Q: What if I need to clone my artificial miRNA or synthetic tasiRNA into a vector with an alternative promoter or selectable marker absent in available B/c vectors?
A: If your expression vector is GATEWAY-compatible, you can use pENTR-AtMIR390a-B/c and pENTR-OsMIR390 for cloning artificial miRNA to use in eudicots and monocots, respectively, and pENTR-AtTAS1c-B/c for cloning synthetic tasiRNA to use in Arabidopsis thaliana and close related species (e.g. Camelina sativa). If you want to clone your artificial miRNA or synthetic tasiRNA into a totally different vector system, you can still use P-SAMS amiRNA Designer or P-SAMS syn-tasiRNA Designer apps to design the artificial miRNA(s) or synthetic tasiRNA(s), respectively, and disregard the information related to the two oligonucleotides compatible with B/c vectors.
Q: Should I design an artificial miRNA or several synthetic tasiRNAs?
A: An artificial miRNA is the preferred option to target a single gene. Artificial miRNA can also be designed to target multiple genes if these share enough sequence similarity. P-SAMS amiRNA Designer outputs the sequence of the two oligonucleotides needed to clone the designed amiRNA into the preferred B/c vector. B/c vectors containing the Arabidopsis thaliana MIR390a or the Oryza sativa MIR390 foldbacks are for use in eudicot or monocots species, respectively.
In Arabidopsis thaliana and other close related species that express miR173 (e.g. Camelina sativa), syn-tasiRNAs can be used to target multiple unrelated genes. For other plant species, co-expression of miR173 together with the syn-tasiRNA construct is necessary to produce syn-tasiRNAs. Several syn-tasiRNAs can be multiplexed in the same construct, with each individual syn-tasiRNA targeting a specific gene or sets of genes. We have successfully tested multiplexing up to two syn-tasiRNAs, but theoretically, more than two syn-tasiRNAs can be multiplexed in the same construct. P-SAMS syn-tasiRNA Designer outputs the sequence of the two oligonucleotides needed to clone the syn-tasiRNA into the preferred B/c vector containing the Arabidopsis thaliana TAS1c precursor.
Q: I already have amiRNA/syn-tasiRNA sequence(s) that I want to use, can I just get the sequences of the two oligonucleotides I need for cloning into the appropriate B/c vector?
A: Yes, for artificial microRNA (amiRNA), use the amiRNA_oligoDesigner.pl tool and for synthetic trans-acting
short-interfering RNA (syn-tasiRNA), use the syntasiRNA_oligoDesigner.pl tool. Both tools are available on
GitHub.
Q: What is the difference between Optimal results and Suboptimal results?
A: The P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer apps output the results in two categories: the Optimal results and the Suboptimal results. The Optimal results category includes designed artificial miRNA(s) or synthetic tasiRNA(s) that target specifically the desired target gene(s). The Suboptimal results category includes artificial miRNA(s) or synthetic tasiRNA(s) that follow the design parameters but that do not target specifically the desired target gene as they have have at least one off-target. An off-target is a gene different than the desired target gene that is predicted to be potentially targeted by the designed suboptimal artificial miRNA.
Q: When designing an artificial microRNA with P-SAMS amiRNA Designer, what should I do if I do not get results?
A: P-SAMS amiRNA Designer will design artificial miRNA(s) that target single genes in most cases. However, when multiple genes need to be targeted simultaneously, P-SAMS amiRNA Designer may not always display results. This is usually due to a limited sequence homology between the selected target genes, or to the presence of off-targets [transcripts with high sequence homology with the desired target transcript(s)] that difficult P-SAMS to design a specific artificial miRNA.
When P-SAMS amiRNA Designer fails to design an artificial miRNA that targets multiple unrelated genes, synthetic tasiRNAs may be a good alternative to use in Arabidopsis thaliana or in other close related species that express the miR173 trigger (e.g. Camelina sativa). Note that for other plant species, co-expression of miR173 together with the synthetic tasiRNA construct is necessary to produce synthetic tasiRNAs. Group your target genes in different groups based on their sequence homology, and have P-SAMS design a syn-tasiRNA for targeting each group of genes. We have successfully tested multiplexing up to two synthetic tasiRNAs (for e.g. for two groups of target genes or two single unrelated genes), but, theoretically, more than two synthetic tasiRNAs could be multiplexed in the same construct.
Q: How many synthetic tasiRNAs can I multiplex in the same construct?
A: We have successfully tested multiplexing up to two syn-tasiRNA in a single construct. But in theory, more than two syn-tasiRNA could be multiplexed. However, it is known that the farther from the miR173 target site the syn-tasiRNA is cloned, the lowest it accumulates in vivo, suggesting that syn-tasiRNAs located too far away from miR173 target site may be very poorly expressed and, consequently, inactive. This may actually limit the total number of effective syn-tasiRNA that can be multiplexed in a single construct. We decided to limit to four the number of syn-tasiRNAs that can be multiplexed in a single construct using P-SAMS syn-tasiRNA Designer.