Multiplexed silencing of <scp>2S</scp> albumin genes in peanut

Abstract

Peanut (Arachis hypogaea L.) is a highly nutritious legume that provides energy-dense food. One obstacle for universal inclusion of peanut products in the diet is the prevalence of peanut food allergy. Although approximately 2% of individuals may become sensitized to peanut and experience an allergic reaction upon ingestion (Gupta et al., 2011), a minor fraction of these reactions may be life-threatening. The 2S albumins (Ara h 2, Ara h 6 and Ara h 7) are the least abundant seed storage proteins but Ara h 2 and 6 are the most potent allergens (Zhuang and Dreskin, 2013). Multiple isoforms of each 2S albumin exist and tightly linked gene copies encode each protein. One copy of each gene spans 83 kb of chromosome Arahy.08 and one extra copy of Ara h 6 is present within a 163-kb segment on Arahy.18 of Arachis hypogaea (Table S1). While previously shown that reduced expression of the 2S albumins, achieved through RNAi knockdown, did not affect seed growth and development, low amounts of protein that could possibly incite an allergic reaction were still detected (Chu et al., 2008). Protein knockouts through induced mutagenesis or discovery of a natural null mutant is an effective way to ensure protein elimination. A null mutant of the β-conglycinin α-subunit gene in soybean was used to backcross breed lines for reduced allergen and improved quality tofu (Song et al., 2014) while a null mutant of the soybean P34 allergenic protein was used in breeding of an improved and low allergen variety (Jeong et al., 2013). CRISPR-Cas gene editing is recognized as an efficient method for creating targeted mutations in multiple alleles and/or genes (Van Eck, 2020). Given the tight linkage of the 2S albumin genes, a multiplex gene editing strategy using CRISPR-Cas9 and two conserved guide RNAs (Figure 1a; Figure S1, Table S2) for each Ara h gene was attempted. We report the successful multiplexed editing of 2S albumin allergen genes of peanut, transmission through the next generation, and protein elimination. Fifty-two out of 113 total Cas9-positive T0 plants recovered using methods described in Supporting Information produced pods of varying seed quality and number. T0 plants were screened for large CRISPR deletions by agarose gel separation of PCR products (Figure 1b; Table S3). Fourteen T0 plants displayed an Ara h 2 deletion with plants in lines 127 and 214 producing seed. Five T0 plants showed Ara h 6 deletions with line 45 producing seed. Twelve T0 plants showed Ara h 7 deletions with line 127 producing seed. Next-generation sequencing of T1 seed/seedlings and Khufu analysis was used to identify smaller CRISPR-Cas9 edits. To maximize the number of T1 individuals screened, each sequencing well combined three individuals and amplicons from all target genes. A large variation in the number of occurrences and changes were identified for the seven Ara h genes. Changes for all Arah6 and Arah2_B were limited while numerous edits for Arah7_A and _B and Ara2_A were identified (Table S4). While focusing on edits which created nearby stop codons, many identified edits were not knockouts as they created in-frame deletions of 3, 6 or 9 nucleotides or nonsense mutations which did not create a nearby stop codon. Sanger sequencing confirmed Ara h CRISPR edits for six T1 individuals as shown in Figure 1, Figure S2 and Tables S5, S6. Individuals without clones for an Ara h gene (nd) may have completely lost the gene which would not be obvious in PCR amplification due to the A and B homoeologs. Clones from line 127, which had both Ara h 2 and Ara h 7 deletions, displayed larger than expected bands which were also cloned and sequenced. These clones showed a 463-bp insertion in Arah2_A and a 206 bp insertion for Arah7_A. The 206 bp Arah7_A insertion was an exact match for the region deleted between the two guides from Arah2_B while the 463 bp Arah2_A insertion had similarity to multiple regions of the Tifrunner. Arahy chromosomes. These complex rearrangements can sometimes occur upon repair of double-strand breaks. Sanger sequenced T1 lines were compared against T0 parents and unsequenced siblings using PCR amplification of the Ara h genes and inheritance of the transgenes using hygromycin resistance gene primers (Figure 1b). Based on amplicon sizes, T1 offspring looked similar to their T0 parents, although larger amplicons were generated for multiple offspring. Offspring derived from lines 214 and 45 seem to have lost the transgenes based on lack of hygromycin resistance gene amplification (Figure 1). Western blots probed with antibodies for Ara h 2 and Ara h 6 confirmed the disappearance of some protein bands (Figure 1c). Protein isolated from line 42 seed lost Arah2_B and retained a wild-type copy of Arah2_A. Protein from multiple sibs of lines 46 and 89 seed and predicted by Sanger sequencing to show knockout of both copies of Ara h 2 was confirmed. Knockouts of Ara h 2 were also found among T2 seed of lines 127, 45 and 214. The protein analysis of Ara h 6 showed offspring of lines 45 and 46 with a reduction, but not a complete absence, of Ara h 6. No antibodies are available for analysis of Ara h 7. The 2S albumin genes in peanut, comprised of seven family members for Ara h 2, Ara h 6 and Ara h 7, were successfully targeted for multiplexed gene editing with a polycistronic tRNA-gRNA (Wolabu et al., 2020) to boost the recovery of mutants. Guides were designed to conserved regions of both A- and B-genome copies of each gene, and although the ability of all guides to cleave the target sequences was tested with an in vitro screen, in vivo efficiency varied: Ara h 7>Ara h 2>Ara h 6. Lines 45 and 46 are particularly promising for elimination of most 2S albumin according to the Sanger sequencing data although the western blot still shows a faint band suggesting some possible residual. More accurate and sensitive methods for quantifying 2S albumins, such as the reverse-phase LC–MS/MS spectral counting used in a previous study (Stevenson et al., 2009), would need to be applied to resolve this question. If elimination is confirmed, a test for reduced allergenicity logically would be conducted with a mouse model or an ex vivo basophil degranulation assay perhaps followed by skin prick assay (Ozias-Akins et al., 2009). Finally, given the identification of unexpected PCR products, whole-genome sequencing of promising lines where the transgene has been genetically removed through segregation would be needed to confirm the changes and identify possible gene rearrangements or off-target genome edits by Cas9. This work was supported by Ukko, Inc. through funding and collaborative discussion. Cassidy McGahee and Huiqin Chen provided technical assistance. The data that supports the findings of this study are available in the supplementary material of this article. Figure S1 Vector plasmid map. Figure S2 Chromatograms of CRISPR-Cas9 edits. Table S1 Positions of Ara h 2, Ara h 6 and Ara h 7 coding sequences on the Arachis hypogaea Tifrunner 2.0 genome. Table S2 Sequences for guide RNAs. Table S3 Primer sequences used for amplification of Cas9 and allergen genes. Table S4 Smaller CRISPR-Cas9 changes among T1 plants detected by NGS and Khufu output. Table S5 Mutations in T1 lines characterized with Sanger sequencing of Ara h 6. Table S6 Mutations in T1 lines characterized with Sanger sequencing of Ara h 7. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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