Background

Programmable genome editing tools derived from clustered regularly interspaced short palindromic repeats (CRISPR) systems have been used to edit a variety of crop genomes [1]. Introduction of gene modifications, such as insertions, deletions, and substitutions, enables rapid trait modification and analysis of gene function. CRISPR/CRISPR-associated protein 9 (Cas9) [2,21]. Despite these merits, genome editing in sorghum has fallen behind that of other cereals because of the difficulty of obtaining appropriate material, such as immature embryos, constraining sorghum tissue culture and stable transformation. Previous reports of CRISPR/Cas9-mediated genome editing in sorghum have described targeting the centromere-specific histone 3 (SbCENH3) gene [22], the α-kafirin gene family [23], the flowering T locus (SbFT), and the gibberellin 2-beta-dioxygenase 5 (SbGA2ox5) gene [24]. Both Agrobacterium-mediated [22] and microprojectile bombardment-derived [25] transformation methods have been developed for sorghum, but research that aims to increase the efficiency of sorghum genome editing, with sgRNA screening as a critical factor to minimize time and effort, is still lacking.

Transient protoplast transfection assays are a versatile, rapid, and high-throughput method for investigating gene expression [26] and subcellular localization of proteins [27] as well as for assessing genome editing efficiency in plants. Reliable protocols involving polyethylene glycol (PEG)-mediated protoplast transfection have been established for various species, such as Arabidopsis thaliana [26], rice (Oryza sativa) [28], and maize (Zea mays) [29]. Protoplast transfection with genome editing tools has been successfully performed, promising highly efficient genome editing not only with Cas9- or Cas12a-mediated systems but also with base editors and prime editors, in lettuce (Lactuca sativa) [30], soybean (Glycine max) [7], petunia (Petunia x hybirda) [31], rapeseed (Brassica napus) [14], and rice [17]. Although sorghum protoplast isolation and transfection have previously been used for plasmid-mediated genome editing [32], here we optimized methods for assessing the editing efficiency and off-target effects of specific sgRNAs for sorghum genome editing with both plasmid-mediated and ribonucleoprotein-based protoplast delivery systems, a necessary step for genetic studies and plant biotechnology.

To improve the precision of genome editing and reduce off-target effects, DNA-free genome editing, in which preassembled Cas9-sgRNA ribonucleoproteins (RNPs) are delivered into protoplasts, has been developed. RNPs can cleave the target region immediately without transcription and translation and are then rapidly degraded, so off-target effects are reduced compared those associated with plasmid-mediated delivery of CRISPR components. RNP systems have been successfully used for genome editing of soybean [7], lettuce [30], petunia [31], wheat (Triticum aestivum) [33], and pepper (Capsicum annuum) [34]. However, RNP-mediated genome editing in sorghum has not yet been reported.

Here, we screened sgRNAs for targeted mutagenesis of four endogenous sorghum genes that are involved with flowering time (FT genes) and vegetative branching (TIL1 gene): SbFT1 (Sb10g003940), SbFT8 (Sb03g034580), SbFT12 (Sb06g012260), and SbTIL1 (Sb06g019010) [35,36,37].

In this study, we present a screening system for precise and highly efficient genome editing in sorghum. We first isolated protoplasts from leaves from sorghum grown under three different cultivation conditions. We analyzed protoplast yield, viability, and transfection efficiency to establish optimal conditions for sgRNA screening. We transfected Cas9-sgRNA expression plasmids into sorghum protoplasts and analyzed the resulting editing efficiencies including that in potential off-target regions by targeted deep sequencing. Furthermore, we tested an RNP system in sorghum protoplasts. Our sgRNA screening system will be a key method for evaluating the activity of sgRNAs for sorghum genome editing.

Methods

Plant material

Commercial grain sorghum (Sorghum bicolor L. cv. Imky1ho) was used in all experiments. Seeds were sown on commercial bed soil. Seedlings were cultivated under three different conditions (Condition 1: 10 days of 16 h light/8 h darkness; Condition 2: 7 days of 16 h light/8 h darkness and 3 days of 24 h darkness; Condition 3: 3 days of 16 h light/8 h darkness and 7 days of 24 h darkness) at 25 ℃.

Protoplast isolation

Protoplasts were isolated using a protocol described previously [38, 39] with the following modifications: 40 young leaves (Fig. 1a) from plants cultivated under each condition described above were cut into 1 cm long pieces, immersed in a 13% mannitol solution, and incubated at 25 ℃ on a shaker with gentle agitation (60 rpm) for 1 h in the dark, after which the solution was exchanged for enzyme solution (Table 1). Using a razor blade, samples were chopped into pieces about 3–4 mm on a side and incubated at 25 ℃ with 60 rpm agitation for 5.5 h in the dark. The digested mixture was filtered through a 70 μm nylon cell strainer and washed with an equal volume of W5 solution (Table 1). The protoplasts were isolated on a sucrose gradient (24%) by swing-out centrifugation at 100 ×g for 7 min. The intact protoplasts were harvested using a Pasteur pipette, after which they were incubated in W5 solution for 1 h at 4 ℃ before being used for the protoplast viability test or protoplast transfection.

Fig. 1
figure 1

Protoplast isolation from leaves from sorghum plants cultivated under three different conditions. a Ten-day-old seedlings grown under different conditions. Condition 1: 16 h light/8 h dark for 10 days; Condition 2: 16 h light/8 h dark for 7 days and continuous darkness for 3 days; Condition 3: 16 h light/8 h dark for 3 days and continuous darkness for 7 days. Scale bars = 1 cm. b Workflow of the protoplast isolation procedure. Enzyme-treated protoplasts were harvested by sucrose gradient centrifugation. c Weight of leaves, obtained following different cultivation conditions, used for protoplast isolation. d Yield of isolated protoplasts from leaves of plants cultivated under each condition. c and d Values (mean ± s.e.m.) were obtained from three independent experiments. One-way ANOVA analysis was applied. ****P < 0.0001; ***P < 0.001; **P < 0.01; ns, not significant (P > 0.05)

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Table 1 Composition of solutions used for sorghum protoplast isolation and transfection
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Protoplast viability test

Evans blue dye solution (0.02%, Sigma-Aldrich) was mixed with an equal volume of sorghum protoplasts in W5 solution and the mixture was incubated at 25 ℃ for 10 min. The numbers of live (unstained) and dead (stained) protoplasts were determined on a hemocytometer under a light microscope. Protoplast viability was calculated as the number of unstained protoplasts / total number of protoplasts.

Guide RNA design

We compared the nucleotide sequences of the target genes with the corresponding reference sequences [SbFT1 (Sb10g003940), SbFT8 (Sb03g034580), SbFT12 (Sb06g012260), and SbTIL1 (Sb06g019010)] using Sanger sequencing (capillary electrophoresis sequencing, Macrogen, Korea) of PCR amplicons (Additional files 1 and 2). Guide RNAs were designed from the analyzed sequences using Cas-Designer [40] with the Sorghum genome database (v1.0). We selected guide RNAs with high microhomology-associated out-of-frame scores with few potential off-targets effects using Cas-offinder [41] with Sorghum genome database (v1.0). Nucleotide alignments were performed using Geneious (version 8.1.9).

Plasmid construction

To construct the pJ4 plasmid (used for sgRNA and Cas9 expression), we changed the initial base of the sgRNA expression module sequence in pBUN421 (Addgene No. 62204) [22,23,24]. Based on former studies, successful plant genome editing requires active sgRNAs and high transformation efficiency. In previous work, sgRNAs designed in silico resulted in editing efficiencies at target genes that differed from the expected result, due to factors such as chromatin accessibility and sequence context. Screening sgRNAs using transient expression in protoplasts is a rapid and stable cell-based method for evaluating their genome editing efficiency. Pre-screening sgRNAs can reduce the number of sgRNAs needed for effectively generating genome-edited plants by transformation. Here, we optimized a sgRNA screening system for precise, efficient sorghum genome editing (Fig. 5). Results from each step of our protocol were verified by multiple tests: determination of protoplast yields from plants grown under three different conditions, protoplast viability and transfection efficiency using Evans blue staining and GFP expression, and the efficiency and precision of editing induced by various sgRNAs by targeted deep sequencing.

Fig. 5
figure 5

Schematic overview of the sgRNA screening system in sorghum. The time required for each step is indicated in parentheses. Plasmids or preassembled RNPs were delivered into sorghum protoplasts, and editing efficiencies were determined by targeted deep sequencing. The whole process can be completed within 16 days

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We found that a period of cultivation in darkness could enhance the protoplast yield and transfection rate. Condition 2 (7 days of 16 h light/8 h darkness and 3 days of 24 h darkness) resulted in a higher yield of protoplasts (1.83 × 106/mL) and more efficient transfection (29.4%) compared to the other conditions (Condition 1: 10 days of 16 h light/8 h darkness; Condition 3: 3 days of 16 h light/8 h darkness and 7 days of 24 h darkness). Cultivation in darkness improved protoplast yield and transfection, but Condition 2 resulted in 2.2-fold higher transfection efficiency than did Condition 3, which included the longest period of darkness. This observation suggests that the length of time in which plants are cultivated in darkness must be optimized to guarantee the highest yield and transfection efficiency of sorghum protoplasts. We recommend optimizing this variable before conducting genome editing experiments, as cell viability and transfection efficiency differ depending on the genotype and the cultivation conditions.

We showed here that CRISPR/Cas9-based genome editing using sgRNAs designed in silico resulted in indel frequencies of up to 77.8% at the target site. Although each sgRNA was associated with a different editing efficiency as measured by the transient protoplast transfection assay, 85% of these sgRNAs showed editing activity in this study. We also found that active sgRNAs induced indels at similar regions in each target gene (sg1 and sg3 in SbFT1; sg2 and sg4 in SbFT8; sg1, sg2, sg4, and sg5 in SbFT12), suggesting the existence of hot spots (Fig. 3). Too few sgRNAs were studied to make a general rule, but we propose that this observation could serve as a clue for sgRNA design and selection in future experiments. Recently a study reported that use of the endogenous U6 promoter (SbU62.3) in Cas9-sgRNA-encoding plasmids increased genome editing efficiency and the homozygous/bi-allelic editing rate compared to the TaU6 promoter, which is widely used in many crop plants [45]. Our guide RNA screening system could be used with this U6 promoter to improve editing efficiency further.

Our study suggests that DNA-free genome editing could also be a valuable tool for sorghum breeding involving genome editing. We succeeded in editing four different genes (SbFT1, SbFT8, SbFT12, and SbTIL1) using Cas9-sgRNA RNPs, observing indel frequencies of up to 18.5%. Relative to plasmid-based delivery systems, RNPs are functional for less time, which can be beneficial for lowering the frequency of off-target effects. Additionally, there is no need to be concerned about transgene integration into the host genome with an RNP system, another advantage when the goal is to improve crop strains. Although the RNP-mediated editing efficiency in this study was lower than that of DNA-mediated genome editing, RNPs could provide an attractive alternative for precise genome editing with a decreased frequency of unintended cleavage sites. The efficiency of protoplast regeneration, an important factor in generating gene-edited plants, is determined mainly by the genotype [46], and it would be very useful to identify the genotype with the highest regeneration efficiency among sorghum cultivars in future studies. An optimized sorghum protoplast regeneration system could be combined with our DNA-free genome editing method to accelerate the generation of transgene-free mutants for practical breeding and the commercial market.

Our sgRNA screening system could also be applied to other genome editing tools. For example, it could be adapted for plant CBE and ABE systems to determine exactly which point mutations would be induced by specific sgRNAs, with the aim of develo** agronomic traits in sorghum. Our system could also be used to verify the possibility of base editor-mediated targeted saturation mutagenesis [47] to generate gain-of-function variants. Furthermore, we plan to use our system to train a machine learning algorithm [48, 49] to generate a scoring system that predicts which target sites would be most amenable to editing in the sorghum genome.

In summary, we have developed a rapid and precise sgRNA screening system for efficient genome editing in sorghum, which can be completed within 16 days. We successfully isolated sorghum protoplasts and edited target genes in them. The protoplast isolation and transfection steps can also be used to study topics such as gene expression and protein localization. In addition, we used our system to verify that Cas9-sgRNA RNPs are an effective genome editing tool in sorghum.

Conclusion

We established an efficient and specific CRISPR/Cas9 screening system for the grain sorghum. This system will allow rapid and precise programmable genome editing in sorghum for crop breeding and plant biotechnology.