DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize

ABSTRACT Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal (ZmPLA1) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger’s sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.

In light of the changing climatic scenario, it is important to accelerate climate-resilient line development.Doubled haploid (DH) technology based on in vivo haploid induction and the successive doubling of haploids is a promising method to develop homozygous lines in 2-3 generations. 1,2,3Thus the DHs can acquire conventional plant breeding to speed up the breeding of high yielding varieties and resistant to the biotic and abiotic stresses.
The successful in vivo maternal haploid induction is triggered by using the inducer line as male, which is a unique mechanism in maize DH production system.CRISPR-Cas9 system has been established as a revolutionary technique with wide applications in plant biology. 4In maize, genome modification has been reported previously for different traits. 5Many quantitative trait loci (QTLs) which significantly affect the haploid induction rate (HIR) have been mapped. 6Among these qhir1 QTL locus has the most significant effect on the haploid induction.
Prigge et al. 7 reported that two key quantitative trait loci, qhir1 and qhir8, lead to high-frequency haploid induction in maize.Fine mapping and sequence analysis studies of these QTLs led to the conclusion that mutations in PHOSPHOLIPASE A1 (ZmPLA1) 8 and DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) 9 have been shown to generate haploids in maize.Both ZmDMP and ZmPLA1 have shown similar expression patterns and subcellular localization in maize.Recent findings reveal that knockout of ZmDMP along with ZmPLA1 triggered haploid induction and exhibited a greater ability to increase the HIR by 5-6 folds. 10here are some newly developed protocols which can be used for the regeneration of the plantlets from the immature embryos of maize, 11 In vitro transcription of the sgRNA, 12 the modified gene gun bombardment technique for an effortless gene transformation 13 and in vitro cleavage of the DNA using CRISPR/Cas9 RNP complex for the pre-validation, functionality and efficiency of CRISPR genome editing system. 14The present study aims at generating mutants for ZmPLA1 gene in maize inbred line of PAU (LM13) through RNP complex-based genome editing to obtain higher HIR.

Materials and Methods
Maize inbred line LM13 (JCY-3-7-1-1-1) was selected to generate the mutants for the target gene, as this line is better responsive to the agronomical practices and a good combiner in hybrid breeding programme.ZmPLA1 gene from the LM13 was cloned, sequenced and aligned to ZmPLA1 locus (GRMZM2G471240) and conserved regions were identified to design sgRNA.The guide RNA was designed using CRISPR Plant v2.0 software (http://crispr.hzau.edu.cn/CRISPR2/) and comparative analysis was done with other software known as CHOP CHOP (http://chopchop.cbu.uib.no/) (Table 1).sgRNA synthesis was followed according to the protocols provided with Thermo Fisher Kit.T7 promotor region was used for the in vitro transcription of sgRNA from its DNA template.in vitro transcribed product purified and maintained for further experiments.Ribo nucleoprotein complex was prepared by taking 2 µg of Truecut Cas9 protein (Thermofisher) and 2 µg of sgRNA at optimal 1:1 concentration and was used for the in vitro cleavage assay and then for the bombardment reactions.in vitro cleavage assay was performed, to check the cleavage efficiency for all these sgRNAs.
Immature embryos were placed on the 0.4 M mannitol containing osmoticum MS medium and used for bombardments.These bombarded embryos were then transferred into the half MS medium to obtain the desired growth of plantlets.Genomic DNA was isolated from the regenerated plantlets using CTAB method. 15The ZmPLA1 gene-specific primers were designed for first exon using Primer 3 software (ZmPLAEX1MDF-5"GTCCATGCAATACCTGTAGC3," ZmPLAEX1MDR-5"AAAGTGGTTGATGTCCTTGG3," ZmPLAEX1MDF1-5"CCATGCAATACCTGTAGCACG3," ZmPLAEX1MDR1-5"GGATGGATGCAAGAACAATGG3").PCR amplified products from all the 288 regenerated plantlets were resolved on 2.5% agarose gel.Cleavage mutation detection assay was performed by GeneArt® genomic cleavage detection kit (ThermoFisher Scientific) to identify the putative mutants.The cleavage detection enzyme T7E1 recognizes and cleaves the heteroduplex loops formed by insertion or deletion of nucleotides.Due to limitations of this assay rest of the remaining samples were sent for the Sanger's sequencing in duplicates.After sequencing, various bioinformatic tools like Chromas software, clustalX 2.0 software, BLAST analysis by NCBI, CRISPR ID and DsDecodedM, were used to align the sequences with reference control (LM13) to identify any mutations like insertion/deletion.The monoallelic and biallelic mutations were detected using the DsDecodedM online web tool.ExPASy and ORF finder were used to track the disturbance in open reading frame or the replacements and rearrangements in amino acids.

Results and Discussion
In the present study, efforts were made to edit the ZmPLA1 locus through site-directed mutagenesis to generate a novel haploid inducer stock in tropical maize background.As previously reported, haploids were produced using crosses to Stock 6 16 and the mechanism of haploid induction was studied later on by three independent laboratories. 8,17,18he mechanism of haploid induction is triggered by a 4 bp mutation in the ZmPLA1 gene 8,18 and further edits in this gene could lead to increase in haploid induction rate of 6-7%.
The sgRNA DNA templates of 120 bp for the sgRNAs namely sg166, sg17 and sg20 were synthesized followed by in vitro transcription that resulted in 100 bp intact CRISPR fragment of sgRNAs (Figs. 1 and 2).Similarly, Hu et al. 19 observed 120 bp of sgRNA DNA template synthesized using four overlapping primers for targeting the EGFR genes.Moreover, Liang et al. 20 transcribed a 100 bp intact fragment of sgRNA from the template while working on isolated cell lines of Jurkat cells.Jinek et al. 21observed cleaved fragments of ≈ 2230 bp and≈3100 bp from 5332 bp intact fragment of human clathrin light chain (CLTA) gene which was an indicator of Cas9 mediated cleavage.Therefore, in vitro cleavage assay was performed on the transcribed gRNAs for determining their cleavage efficiency.The assay resulted in two cleaved bands of size≈210 bp and≈165 bp and≈230 and≈150 bp in case of sg166 and sg17 respectively.However, the sgRNA sg20 resulted in an intact band of 376 bp depicting no proper cleavage (Supp Fig. 3); hence the sgRNA sg166 and sg17 were selected for further study.
6][27] However, immature embryos are the material of choice in maize for the efficient production of transgenic lines through particle bombardment or Agrobacterium transformation. 28he advantages of bombardment over Agrobacterium mediated transformation were studied by Mookkan. 13In the present study, a total of 2315 embryos were bombarded using both the sgRNAs and 373 plants were regenerated and survived upto hardening.Malini et al. 29 reported that the concentrations of BAP (0.5, 1.0 and 1.5 mg/l), NAA (0.1, 0.2 and 0.3 mg/l) and kinetin (1.0 mg/l) to be used for plant regeneration in case of immature embryos.Whereas Guruprasad et al. 28 regenerated plantlets from immature embryos using MS medium formulations.
The cleavage detection assay was performed on the few samples to detect the mutations in the targeted region along with LM13 (Nontransformed) as a control.The cleaved bands in the positive control validated the efficiency of the sgRNAs; meanwhile, the single band in negative control LM13 against the two cleaved bands in the sample no.102 showed the presence of mutation.All the edited plants (288) obtained were further validated through Sanger's Sequencing.The sequences obtained were aligned with the LM13 sequence (as reference) using ClustalX 2.0.Mutations were identified in only three samples after alignment of the sequences.Two sequences were found to have mutations just near the target site of sgRNA and only one sequence showed one nucleotide of substitution and deletion at the target site of sgRNA.The results of the BLAST for the edited plant no.258, 287 and 21 are given in Supp Fig 4, Figs. 5 , and 6.In case of a diploid organism, it is very important to disrupt both the copies of a gene to suppress its expression.Homozygous biallelic mutant for the plant no.21 has the substitution in both the alleles as depicted in Supp Fig. 7.The monoallelic mutant in the plant no.258 has one base substitution in a single allele as represented in Fig. 8.
At the protein level, the ExPASy tool had shown the disruption of the 33 amino acids chain in the putative mutant sample no.287.These results were cross-checked with the ORF finder online web tool.The putative sample was then self-pollinated to obtain T 1 seeds from the edited T 0 mutant.A single cob containing 48 seeds was obtained from this mutated plant.These T 1 edited seeds are being maintained for the generation of T 2 progeny and further needs screening to evaluate the haploid induction ability by crossing with different maize inbred lines.
Although the undesirable effects coupled with backcrossing and other conventional methods are reduced using biolistic delivery of RNP complexes into plant cells.Current methods of genetic transformation together with low efficiency of DNA delivery into target regions and less regeneration rate of plants create hindrance for editing plant genomes.Feng et al. 30 targeted a marker gene Zmzb7 for genome editing which is responsible for leaf coloration in plants.Wang et al. 31 edited the ZmLG1 gene, which is a major determinant of leaf angle in maize.The loss of function of this gene resulted in a reduction of leaf angle due to the absence of auricle and ligules.Our research shows successful utilization of ZmPLA1 gene for inducing haploid induction in tropical maize line.There are recent studies based on the identification of genes such as ZmDMP 9 and ZmPLD3 10 that are responsible for haploid induction.
In-vitro cleavage assay can be successfully used to pre-evaluate the efficiency of the designed sgRNA's in the CRISPR Cas9 genome editing experiments.Immature embryos about 12-14 days after pollination can be successfully used for the gene gun bombardment via ribonucleoprotein complex delivery.Disruption of one of the 33 AA protein frame in first exon shows possibility of haploid induction.