Robust CRISPR/Cas9 mediated genome editing and its application in manipulating plant height in the first generation of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro)

Bamboo covers over 30 million hectares of land across the world, and around 2.5 billion people are directly producing and consuming bamboo. However, probably it is the only major agronomic crop that is nearly impossible to make improvement by traditional breeding as the flowering time of most bamboos exceeds 70 years. Bamboo is the fastest growing plant on this planet, whearas, our knowledge on the underlying mechanisms is quite limited mainly due to the lack of efficient genetic manipulation tools. Therefore, the application of genetic engineering technology on bamboo is extremely important for its breeding and basic research. CRISPR (Clustered Regularly Interspaced Short Palindromic repeat)/Cas9 system provides straight forward ways for genome editing in many plants, but was never used in bamboo. In this study, we optimized the CRISPR/Cas9 elements in bamboo protoplast, and for the first time we established an efficient protocol for artificially producing the hexaploid bamboo mutant in its first generation by simultaneously targeting all homo-alleles or specifically targeting one allele of a putative phytoene synthase gene through CRISPR/Cas9 technology. Furthermore, we produced a bamboo mutant with increased plant height by knocking out one Gibberelline-responsive gene that we identified previously, which provides a good material for the future study on the molecular mechanism that controls the fast growth of bamboo.

showed strong signals within 72 h, indicating that the mGFP function was restored by the CRISPR/Cas9 system through deleting the additional 'guanine' ( Figure 1C). The OsU6a and OsU6c promoters work as well, however, with lower efficiency than the OsU6b promoter, as positive signals were only occasionally observed with more than 10 replicates. Taking together, the UBI-Cas9/OsU6b-sgRNA construct effectively works in bamboo protoplast and was used for the following endogenous gene editing in Ma bamboo.
The putative phytoene synthase (PSY1) in bamboo, whose homolog in maize functions in carotenoid biosynthesis (Zhu et al., 2016), was selected for the initial test. Three bamboo PSY1 alleles (DlmPSY1-A, DlmPSY1-B and DlmPSY1-C) were identified and cloned by a homology cloning strategy (Figure 1D). To mutate all copies of DlmPSY1, sgRNA1 targeting a conserved site among all DlmPSY1 loci was designed (Figure 1D). In addition, the sgRNA2 target site containing 2-3 single-nucleotide polymorphisms (SNPs) in the spacer region among three DlmPSY1 homoeoalleles was selected to test the tolerance of sgRNA mismatches ( Figure 1D).
A total of 1600 bamboo calluses induced from stem were transformed as described previously (Ye et al., 2017). In total, 34 independent transgenic lines were confirmed positive (2.1%) by PCR. Based on Sanger sequencing results, 22 (100%) and 10 (83.3%) independent T0 lines were edited in the sgRNA1 and sgRNA2 regions, respectively ( Figure 1E), indicating that both constructs effectively induce endogenous gene editing.
The editing profiles were further analysed by sequencing. Eighteen lines (81.8%) contained putative homozygote/biallelic mutations in all subgenomes at the sgRNA1 target site. In some lines, putative homozygote/biallelic mutations exist in one subgenome, while heterozygote or chimeric mutations appear in other subgenomes (T0-10 and T0-26; Figure 1F). Eight mutation types were identified from 590 independent clones ( Figure 1G). The most frequent mutation type was deletion (75%), of which 59.1% are small deletions (<2 bp). The ratios of large fragment deletions (≥14 bp), insertions and combined indels were 15.9%, 2.21% and 7.82%, respectively ( Figure 1G). Since bamboo propagates through asexual budding, those homozygote/biallelic mutations will remain in the genome of their offspring clones during breeding.
Eighteen lines (81.8%) with homozygote/biallelic mutations in all subgenomes at the sgRNA1 site exhibited albino phenotypes ( Figure 1J), which appeared at an early stage during tissue culture and persisted at the plantlets stage ( Figure 1J). Those results suggest that genome editing takes place at an early stage in embryonic cells and led to the loss of function of all DlmPSY1 alleles. Similar results were reported in rice, wheat or cotton (Wang et al., 2014;Wang et al., 2018;Zhang et al., 2014). In case of sgRNA2, although DlmPSY1-A was mutated, no visible phenotypic change was observed due to the existence of the wild-type DlmPSY1-B and DlmPSY1-C alleles.
Next, we applied this technology in bamboo molecular research. Bamboo is the tallest grass in the world, while the underlying mechanism is unknown. Previously, we identified several Gibberellin-responsive genes including GRG1 (GA-responsive gene 1, PH01004823G0070) that potentially acts in controlling bamboo height . Here, two homozygote grg1 mutants (efficiency 40%) in Ma bamboo were produced using our optimized CRISPR/Cas9 technology. Mutation in GRG1 increased plant height ( Figure 1K), mostly due to elongated internodes ( Figure 1L-N). Sequencing results confirmed that the grg1 mutant has the putative homozygous mutation in A1 subgenome, biallelic mutation in B1 subgenome and homozygous mutation in C1 subgenome ( Figure 1O), indicating the loss of function of GRG1 in transgenic bamboo. To our knowledge, this is the first example on controlling bamboo height through gene manipulation, which will contribute to subsequent studies on the molecular mechanisms behind the fast growth of bamboo.
In summary, for the first time we engineered the hexaploid Ma bamboo through CRISPR/Cas9 technology. The homozygote mutations were obtained in the first generation of transgenic lines, which are extremely important for bamboo species due to its long vegetative growth periods. We also confirmed the albino phenotype of dlmpsy1 mutant in bamboo and generated a bamboo mutant with altered plant height. This demonstrates the applicability of CRISPR/Cas9 in bamboo and thereby boosts future bamboo research and breeding.