Update:
1) Improving the efficiency of CRISPR/Cas9 genome editing. We harvested seed (T1 generation) from 48 T0 generation plants that were transformed with the new system named pSoy2-inCas9. We are currently testing 24 T1 seedlings from each of the T0 plants for the presence of edits in the soybean FAD2A and FAD2B genes.
We are also continuing to follow inheritance of intron Cas9 and mutations induced by it from the original version of the construct. T2 seedlings from one event are being screened to identify lines that are homozygous for FAD2A and FAD2B mutations and lack the intron Cas9 construct. We also tested T1 seeds from a second T0 plant transformed with this construct and found that there were no edits in the FAD2A and FAD2B genes, so this line will not be further pursued. We expect differences from one transgenic line to another, so it is not unexpected that the second line could not produce edits. This result further highlights the advantages of the newly improved pSoy2-inCas9 system, because we are now able to generate more T0 lines, and so, there is a higher probability of recovering multiple lines that are able to produce the gene edits that are of interest.
2) Testing CRISPR/Cas9 base editors in soybean. We previously reported that the transgenic lines carrying the first version of base editors did not produce T1 progeny that carried base edits. We were going to take these to the T2 generation and test for the presence of edits, but decided to stop work with these lines after the seeds were harvested. The success of the improved transformation vector coupled with the introduction of the intron in the pSoy2-inCas9 system caused us to rethink the strategy. A new set of transgenic plants that applies the features of the pSoy2-inCas9 system to base editing is being produced. We expect that we will be able to recover many more transgenic lines with more active base editors, and so, the probability of recovering T1 plants with the desired base edits will be higher.
We have modified the CRISPR-Cas9 system to make it more efficient for use in soybean. This was accomplished in two steps. The first was that the process of soybean transformation was significantly improved by making regeneration of transgenic plants more efficient. One of the challenges of soybean transformation is that usually only a few plants are recovered that carry the desired transgene. The improved system allows us to recover many more transgenic plants, which means that we increase the likelihood of recovering plants that not only carry the desired transgene, but also produces more lines to choose from in order to select the best one(s). The second improvement was the addition of an intron sequence into the Cas9 gene. This enabled us to increase the frequency of edits induced by the CRISPR-Cas9 system in the offspring of the transgenic lines. These improvements are now being extended to base editing and other CRISPR-Cas applications in soybean.