The CRISPR/Cas9 system has been widely applied for plant genome editing. The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif (PAM) sequences in the canonical form NGG and non-canonical NAG. Although these PAM sequences are extensively distributed across plant genomes, a broader scope of PAM sequence is required to expand the range of genome editing. Here we report the adoption of three variant enzymes, xCas9, SpCas9-NG and XNG-Cas9, to produce targeted mutation in soybean. Sequencing results determined that xCas9 with the NGG and KGA (contains TGA and GGA) PAMs successfully induces genome editing in soybean genome. SpCas9-NG could recognize NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT PAM sites. In addition, XNG-Cas9 was observed to cleave soybean genomic regions with NGG, GAA and AGY (contains AGC and AGT) PAM. Moreover, off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9 (enhanced specificity SpCas9) and exCas9 (enhanced specificity xCas9) could improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency. These findings significantly expand the scope of Cas9-mediated genome editing in soybean.
Genetic mutation enables reverse genetics and identification of gene functions in plants. Several methods including physical, chemical and biological (e.g., T-DNA/transposon insertion) mutageneses have been widely applied for characterizing gene function in model plants, such as Arabidopsis (Alonso et al. 2003) and rice (Yang et al. 2013). However, random mutagenesis is time-consuming and laborious and many undesirable mutations can also be induced during these processes. Therefore, new technologies regarding targeted mutagenesis are required for basic plant research and crop improvement. The emergence of programmable sequence-specific nucleases (SSNs) provides a prospect for targeted mutation. SSNs are able to generate double-strand DNA breaks (DSBs) at given genomic sites which will be repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair pathway which often produces nucleotide substitutions, insertions and deletions (indels) at the repaired sites. HDR can repair the DSBs if undamaged homologous templates are available at the time (Symington and Gautier 2011).
The recently-characterized clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) has been successfully used to generate site-specific DSBs for genome editing in numerous organisms including plants (Doudna and Charpentier 2014; Feng et al. 2013; Shan et al. 2013; Yin et al. 2017). It uses base-pairing RNA to guide Cas9 endonuclease to targets where the enzyme cleaves DNA in a sequence-specific manner (Hsu et al. 2014). Target site specificity is strictly determined by a chimeric single guide RNA (sgRNA) and a short trinucleotide protospacer adjacent motif (PAM) in the genome (Anders et al. 2014; Sternberg et al. 2014). Therefore, this PAM requirement greatly reduces the targetable sites of CRISPR/Cas9 system in the genome. Moreover, some applications of the CRISPR system, such as precision editing through homology-directed repair (Paquet et al. 2016), CRISPR interference (CRISPRi) (Qi et al. 2013), CRISPR activation (CRISPRa) (Gilbert et al. 2013) and base editing (Komor et al. 2016) are even more severely restricted by the PAM. So far, the commonly used SpCas9 in rice has been shown to recognize PAM sequences in the canonical form NGG and non-canonical NAG (Meng et al. 2018).
Although these PAM sequences are widely distributed across plant genomes, a broader scope of PAM sequence is still needed to expand the range of genome editing. To address this limitation, researchers have exploited the natural diversity of the CRISPR-Cas system and several new Cas proteins, including SaCas9 (Hua et al. 2019b; Kaya et al. 2016; Qin et al. 2019) and Cas12a (or Cpf1) (Endo et al. 2016; Tang et al. 2017; Wang et al. 2017), have been widely used in plants. Recently, a new SpCas9 variant named xCas9 was generated by a phage-assisted continuous evolution experiment, and the laboratory-evolved protein that was produced was able to recognize a broad range of PAM sequences including NG, GAA and GAT in human cells (Hu et al. 2018). This xCas9 variant has been reported to be able to recognize a variety of PAMs including NG and GAA in rice, but its editing efficiency was relatively low (Wang et al. 2019; Zeng et al. 2020). Guiding by the structure of the SpCas9-sgRNA-DNA complex, a rationally engineered SpCas9 variant (SpCa9-NG) able to recognize relaxed NG PAMs was developed (Nishimasu et al. 2018). Compared with xCas9, SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs in rice (Endo et al. 2019; Hua et al. 2019a; Ren et al. 2019; Zeng et al. 2020). Nevertheless, both xCas9 and SpCas9-NG expanded the scope of genome editing with a reduced efficiency in Arabidopsis thaliana (Ge et al. 2019). In tomato, xCas9 could not recognize NG PAMs, and SpCas9-NG was only functional in some NG PAMs tested (Niu et al. 2020). Interestingly, XNG-Cas9, an xCas9 and the Cas9-NG hybrid, presented the broadest target scope compared with SpCas9, xCas9 and SpCas9-NG for genome editing in tomato and Arabidopsis (Niu et al. 2020). However, a systematic analysis of genome editing for those three Cas9 variants in soybean (Glycine max (L.) Merr.) is still lacking. Soybean is an economically important oil and protein crop, and SpCas9 has been shown to function successfully for targeted mutagenesis of the soybean genome (Cai et al. 2015; Jacobs et al. 2015; Sun et al. 2015; Tang et al. 2016). Moreover, mutations induced by SpCas9 can be faithfully transmitted to the next generation in soybean (Bai et al. 2020; Cai et al. 2018).
In this study, we report the adoption of three variant enzymes, xCas9, SpCas9-NG and XNG-Cas9, to produce targeted mutation in soybean. Sequencing results proved that xCas9 successfully produced mutations in genomic regions with PAM sequences containing NGG and KGA (contains TGA and GGA). SpCas9-NG had the broadest range of PAM sequences among the three Cas9 variants, and it could recognize NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT PAM sites. For XNG-Cas9, it had robust editing activity on NGG, GAA and AGY (contains AGC and AGT) PAMs in soybean. Moreover, both SpCas9 and xCas9 caused off-target editing events at GGA PAM sites, and two high-fidelity Cas9 variants including eSpCas9 (enhanced specificity SpCas9) and exCas9 (enhanced specificity xCas9) were generated. We found that both eSpCas9 and exCas9 could improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency in soybean.
Gene editing in soybean by xCas9
We first generated xCas9 3.7, whose mutations include A262T, R324L, S409I, E480K, E543D, M694I and E1219V, through mutating SpCas9 as described (Hu et al. 2018). Then xCas9 3.7 was fused to a 3 × FLAG-tag and the nuclear localization signals (NLSs). This fusion protein was cloned into a binary vector under the control of a double 35S promoter. The vector also contained a sgRNA driven by a soybean U6 promoter, leading to the vector pGmU6-xCas9 (Fig. 1A). A similar strategy was applied to generate the vector pGmU6-SpCas9 (Fig. 1A). To test the endogenous genome editing capability of this evolved Cas9 variant in soybean, four types of PAM sites including NG (contains CGG, AGA, TGA, CGA, GGA, AGC, TGC, CGC, GGC, AGT, TGT, CGT and GGT), GAA, GAT and NAG were targeted, and three independent genomic loci were selected for each PAM. In total, 19 genes were selected as target genes: miR156a, miR156c, miR156f, miR166a, miR167a, miR172a, miR172b, miR172c, miR172d, miR2118a, miR396a, miR396c, miR396e, miR397a, miR398a, miR399d, miR408a, FEI and NARK. We designed 48 sgRNAs that target these genes, and 12–17 independent soybean transgenic hairy roots were generated for each sgRNA (Table S1).
The editing efficiency of xCas9 on canonical PAM NGG was studied first, and deep amplicon sequencing results showed that xCas9 achieved a mutation efficiency of 20.39% in the tested soybean transgenic hairy roots, which is comparable to SpCas9 (18.3%, P value = 0.71, two-sample independent t test) (Fig. 1B and C). Interestingly, it has been reported that the editing efficiency of xCas9 for NGG PAM is quite low in a number of plant species (Ge et al. 2019; Ren et al. 2019; Zeng et al. 2020). However, we found that xCas9 exhibits higher mutation efficiency than SpCas9 at the CGG site, but this difference is not significant, which might be caused by the species difference.
Considering that SpCas9 cleaves targets with NAG PAM at lower efficiency than NGG PAM (Hsu et al. 2013), we next investigated editing efficiency of SpCas9 and xCas9 with NAG PAM. Three sgRNAs, designed separately to target NARK, miR396e and miR156a, were applied and the mutation rate of SpCas9 was 2.34% at the CAG PAM site, whereas an extremely low mutation efficiency (0.24%) was detected in hairy roots with xCas9 (Fig. 1B). These results indicate that xCas9 is potentially efficiently functional on soybean genomic regions with NGG PAM not NAG PAM.
Second, we tested the editing efficiencies of both xCas9 and SpCas9 on non-canonical PAM GAA and GAT. Unfortunately, neither xCas9 nor SpCas9 were able to effectively create mutation at sites with GAA and GAT PAMs (Fig. 1B), which is consistent with the earlier finding that xCas9 could not recognize GAA and GAT PAMs in tomato (Niu et al. 2020). Subsequently, for non-canonical PAM sequence NG including TGA and GGA, as expected, xCas9 worked well whilst SpCas9 did not function efficiently at the sites tested. Although xCas9 exhibited mutation rates of 10.45% and 10.06% for TGA and GGA, respectively, deep amplicon sequencing from 86 transgenic soybean hairy roots showed that xCas9 generated targeted mutagenesis with high efficiency in NARK (27.22% and 29.23% for TGA and GGA, respectively) but low efficiency in other tested genomic loci including miR156a (0.18%), miR396c (0.09%), miR396a (0.23%) and miR156c (0.05%) (Fig. 1B and C). Moreover, xCas9 induced mutations at some NGC and NGT PAM sites, but the mutation efficiencies varied at different target sites (Fig. 1B, C and Table S1). These observations indicate that different genomic loci have different effects on the editing efficiency of xCas9, which are consistent with earlier findings in rice (Hua et al. 2019a, Zeng et al. 2020). In addition, both xCas9 and SpCas9 could not efficiently cause mutation in genomic regions with NG PAMs excluding TGA and GGA PAMs. Collectively, these results showed that xCas9 has robust editing activity on NGG and KGA PAM in soybean.
Gene editing in soybean by SpCas9-NG
We introduced the L1111R, D1135V, G1218R, E1219F, A1322R, R1335V and T1337R mutations into SpCas9 to generate the SpCas9-NG variant, and applied it to replace xCas9 in the vector pGmU6-xCas9, leading to the vector pGmU6-SpCas9-NG (Fig. 2A). Then, the editing efficiencies of SpCas9-NG and SpCas9 were compared on various PAMs that were studied in xCas9. Here we found that SpCas9-NG (7.03%) had a significantly lower (P value = 0.02, two-sample independent t test) editing efficiency than SpCas9 (18.3%) at the CGG PAM site in soybean (Fig. 2B and D), which is consistent with the finding in mammalian cells (Nishimasu et al. 2018). At the GAA and GAT PAM sites, SpCas9-NG showed robust editing activity, with indel mutation frequencies of 10.09% (GAA) and 2.71% (GAT) (Fig. 2B and D). For NGA and NGT PAM sequences, SpCas9-NG (5.32% for NGA; 8.89% for NGT) was more efficient compared with SpCas9 (0.98% and 0.48% for NGA and NGT, respectively) (Fig. 2C and D). In addition, SpCas9-NG could be functional on RGC PAM, and its indel mutation rate was 5.42% and 1.84% for AGC and GGC, respectively (Fig. 2B and D). Collectively, our results showed that SpCas9-NG recognizes a broad range of PAM sequences in soybean including NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT.
Gene editing in soybean by XNG-Cas9
Recently XNG-Cas9, an xCas9 and Cas9-NG hybrid, was shown to efficiently-mutagenize endogenous target sites with NG, GAG, GAA and GAT PAMs in the tomato and Arabidopsis genomes (Niu et al. 2020). The XNG-Cas9 was generated by combining xCas9 mutations with SpCas9-NG mutations (A262T, R324L, S409I, E480K, E543D, M694I, L1111R, D1135V, G1218R, E1219F, A1322R, R1335V and T1337R), and we used it to replace the xCas9 in the vector pGmU6-xCas9, leading to the vector pGmU6-XNG-Cas9 (Fig. 3A). Next, we assessed the editing efficiency of XNG-Cas9 on the above PAM sites in soybean. Compared with the 18.3% mutation efficiency of SpCas9 at the CGG site, XNG-Cas9 only achieved a 2.58% mutation efficiency, which is also significantly lower than SpCas9-NG (P value = 0.03, two-sample independent t test) (Fig. 3B and C). At the GAA PAM sequence, XNG-Cas9 showed robust editing activity, with an indel mutation frequency of 9.63% (Fig. 3B and C), which is comparable to SpCas9-NG (P value = 0.92, two-sample independent t test). For the AGY (contains AGC and AGT) PAM site, XNG-Cas9 (3.91% for AGC; 4.81% for AGT) had significantly higher editing efficiency than SpCas9 (P value = 0.008 for AGC; P value = 0.0001 for AGT, two-sample independent t test) (Fig. 3B and C). However, XNG-Cas9 could not efficiently cause indel mutations on other tested PAM sequences. Collectively, these results indicate that XNG-Cas9 has a robust editing activity at the NGG, GAA and AGY PAM in soybean.
Gene editing in soybean by eSpCas9 and exCas9
Understanding the scope of off-target mutations in CRISPR-edited plants is critical for research, and SpCas9 has been reported to induce highly specific genome editing in rice (Tang et al. 2018). Here the specificity of SpCas9 was evaluated in soybean. We examined the off-target editing possibilities of SpCas9 on sgRNAs targeted GmNARK locus which presented relatively high editing efficiency. The top five PAM sites (contains CGG, CAG, AGA, TGA and GGA) with the highest editing efficiency in GmNARK locus were studied using the online tool CRISPR-GE (Xie et al. 2017) to predict potential off-target sites of these sgRNAs. With the exception of sgRNA with the PAM sequence GGA, sequencing of these predicted potential off-target sites with one to three mismatches to the sgRNAs did not reveal any editing events (Table 1). However, where a single mismatch was present inside a 19 nt guide sequence of the sgRNA with the GGA PAM site, off-target editing was observed in pGmU6-SpCas9. The off-target effect of this sgRNA was investigated for pGmU6-xCas9 and it showed the highest on-target gene editing efficiency among pGmU6-SpCas9, pGmU6-xCas9, pGmU6-SpCas9-NG and pGmU6-XNG-Cas9. We observed that pGmU6-xCas9 could generate more edited transgenic hairy roots than pGmU6-SpCas9 at off-target sites (Table 1). To improve specificities of both SpCas9 and xCas9, we generated an enhanced specificity SpCas9 (eSpCas9) and exCas9 according to a previous study (Slaymaker et al. 2016). The K848A, K1003A and R1060A mutations were introduced into SpCas9 and xCas9 to generate the eSpCas9 and exCas9 variants, respectively, leading to the vectors pGmU6-eSpCas9 and pGmU6-exCas9 (Fig. 4A). Next, we assessed the on-target and off-target editing efficiencies of both eSpCas9 and exCas9 for this sgRNA with the GGA PAM site. Deep amplicon sequencing results showed that eSpCas9 achieved an indel mutation frequency of 9.96% in GmNARK target, which is significantly higher than SpCas9 (Fig. 4B and C) (P value = 0.03, two-sample independent t test). However, the off-target editing efficiency of eSpCas9 was 1.07%, which is significantly lower than SpCas9 (3.52%, P value = 0.02, two-sample independent t test) (Fig. 4B). For exCas9, on-target editing efficiency was lower than xCas9, but this difference was not significant (Fig. 4B and C) (P value = 0.11, two-sample independent t test). Nevertheless, exCas9 showed an off-target editing efficiency that was significantly lower than xCas9 (0.74% for exCas9; 19.27% for xCas9, P value = 0.003, two-sample independent t test) (Fig. 4B). Together these results indicate that both eSpCas9 and exCas9 can improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency.
Although SpCas9 has been successfully applied in soybean genome editing, the NGG PAM requirement of canonical SpCas9 would limit its targeting scope. For example, for functional knockout of the miRNA in soybean, the bioinformatic analysis shows that 590 out of 756 miRNAs can be targeted for their mature miRNA regions by SpCas9, nevertheless, mature miRNA regions of all miRNAs can be targeted by SpCas9-NG (Table S2), supporting that Cas9 with the expanded PAM compatibility is able to broaden the genome targeting range in soybean.
Several amino acids changes in SpCas9 can produce a series of variants of the SpCas9, which have the broader PAM compatibilities. However, it is not clear that the effect of these protein sequence changes on DNA repair outcomes following Cas9 activity in plant. Analysis of DNA repair outcomes through comparing the sizes of indels induced by SpCas9 and its three variants at the target genomic sites, we found that more than 95% of indels is deletion (Fig. S1). Approximately a quarter of deletions induced by SpCas9 is 4 bp, xCas9 produced about 25% of deletions with 2 bp, SpCas9-NG generated 16.32% of deletions with 5 bp, and nearly 50% of deletions produced by XNG-Cas9 is 7 bp (Fig. S1), suggesting that different Cas9 proteins can produce different DSB repair outcomes in soybean.
In summary, we generated three Cas9 variants including xCas9, SpCas9-NG and XNG-Cas9, and demonstrated that they recognized various NG PAMs in soybean. Moreover, SpCas9-NG had the broadest range of PAM sequences among the three Cas9 variants. In addition, two high-fidelity Cas9 variants including eSpCas9 and exCas9 significantly improved the specificity of GGA PAM sequence recognition without reducing on-target editing efficiency in soybean. These findings expand the scope of Cas9-mediated genome editing in soybean.
Materials and methods
The xCas9 (xCas9 3.7), SpCas9-NG, XNG-Cas9, eSpCas9 and exCas9 used in this study were generated through mutating SpCas9 according to previous studies (Hu et al. 2018; Nishimasu et al. 2018; Niu et al. 2020; Slaymaker et al. 2016). The SV40 nuclear localization signal (NLS) and nucleoplasmin NLS were then added to the N- and C-terminus of the xCas9. Then xCas9 with the NLS was cloned into the pCAMBIA1300 backbone vector using BglII and BamHI. The bar gene, with a doubled 35S promoter, conferring resistance to the herbicide bialaphos was inserted behind the xCas9 cassette. In addition, a DNA fragment comprised of the GmU6 promoter, two BsaI sites and the sgRNA scaffold of SpCas9 was produced by overlap PCR. The GmU6 promoter sequence was cloned from the genomic DNA of soybean variety Williams 82. Finally, the resultant DNA fragment was cloned upstream of the xCas9 cassette after HindIII and XmaI digestion, leading to the vector pGmU6-xCas9. The same strategy was applied to generate the vector pGmU6-SpCas9, pGmU6-SpCas9-NG, pGmU6-XNG-Cas9, pGmU6-eSpCas9 and pGmU6-exCas9. The sgRNAs were designed using the CRISPR-GE tool (Xie et al. 2017), and the 19 bp target sequence of sgRNA was amplified by PCR. Annealed oligo adaptors were inserted into the BsaI-digested pGmU6-SpCas9, pGmU6-xCas9, pGmU6-SpCas9-NG, pGmU6-XNG-Cas9, pGmU6-eSpCas9 and pGmU6-exCas9 vectors. Sequencing confirmed the fidelity of the vectors. Primers used for vector and sgRNA constructions are listed in Table S3.
Agrobacterium rhizogenes-mediated transformation of soybean hairy roots
Seeds of soybean cultivar Williams 82 were grown in pots in a growth room at 28 °C with a 16-h light/8-h dark cycle. Cotyledons of Agrobacterium rhizogenes strain K599 were transformed with the range of vectors (Wei et al. 2017). In brief, the K599 strain-harbouring construct was injected into hypocotyls of 5-day-old soybean seedlings and then given 2–3 weeks of additional growth for generation of transgenic hairy roots. DNA was prepared from hairy roots and PCR reactions with specific primers specifically targeted sgRNA. Only DNA samples confirmed by PCR were kept for further analysis.
Deep amplicon sequencing and data analysis
Genomic DNAs were prepared from transgenic soybean hairy roots and PCR amplicons of each targeted locus were sequenced. For SpCas9 and xCas9, primers containing barcode sequences were applied in PCR reactions, and the resultant PCR products were sequenced on an Ion Torrent PGM platform. For SpCas9-NG and XNG-Cas9, two rounds of PCR were performed (Liu et al. 2019), and the resultant products were sequenced on an Illumina HiSeqX instrument. This PCR strategy was also used for off-target analysis. Primers used for the target amplification are listed in Table S3.
Indel frequencies were quantified according to an earlier study with small modifications (Komor et al. 2016). In brief, sequencing reads of individual amplicons were separated by searching matched primers. A 33-bp window of reference sequence was used to check the potential indels caused by CRISPR. This 33-bp indel window includes the 3-bp PAM sequence and its 28-bp upstream flanking sequence as well as 2-bp downstream flanking sequence. For each sequencing read, we first identified the 33-bp indel window by scanning the exact matches to two 15-bp sequences that flank both sides of this indel window. If exact matches were located, reads were kept for subsequent analyses. If the length of the indel window exactly matched the reference sequence, then the sequencing read was classified as not containing an indel. If the length of the indel window was two or more bases longer or shorter than the reference sequence, then the sequencing read was classified as an insertion or deletion, respectively.
Predictions of off-target sites were determined by the online tool CRISPR-GE (Xie et al. 2017). Homologous sequences with up to 3 bp mismatches to target sites were listed as potential off-target sites (Table 1). For five PAM sites with the highest editing efficiency in GmNARK locus, these potential off-target sites were amplified from transgenic hairy roots for Sanger sequencing. Then, deep amplicon sequencing was used to study the specificity of the GGA PAM sequence. Primers for off-target amplification are listed in Table S3.
Deep sequencing data produced in this article have been submitted to NCBI under accession number PRJNA685266.
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This work was supported by the grants from Natural Science Foundation of Jiangxi Province (20171ACB20001) and National Natural Science Foundation of China (31800224, 31960138 and 31960433).
Conflict of interest
The authors have no conflicts of interest to declare.
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He, R., Zhang, P., Yan, Y. et al. Expanding the range of CRISPR/Cas9-directed genome editing in soybean. aBIOTECH (2021). https://doi.org/10.1007/s42994-021-00051-4
- Genome editing
- Protospacer adjacent motif