Preliminary Study On Rubber Tree Gene Editing By CRISPR/Cas9 Technology

Natural rubber is an important strategic material and industrial raw material worldwide.As the only commercial source of natural rubber,Brazilian rubber tree(Hevea brasiliensis Muell.Arg.)is of high economic value.There has been great interest in using classical breeding approaches in rubber tree to improve latex yield and quality,along with disease and stress resistance.However,being a cross-pollinated perennial tree species with a long juvenile phase,genetic improvement of rubber tree through conventional breeding is troublesome and time-consuming.Therefore,more efficient molecular methods are urgently needed to accelerate the trait improvement of rubber tree.Recently,CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9),a powerful genome editing tool,has achieved site-directed mutagenesis of genes in many plant species.However,the application of CRISPR/Cas9 in rubber tree has not yet been reported.Here,we describe the efficient targeted mutagenesis in rubber tree.By transformation of a plasmid expressing CRISPR/Cas9 genome editing components,as well as direct delivery of CRISPR/Cas9 ribonucleoproteins(RNPs),targeted mutagenesis were successfully induced at several phosphatidyl ethanolamine-binding protein(PEBP)genes.Precise manipulations of these flowering-regulating factors could help modulate the vegetative and reproductive growth in rubber tree,and thus to increase yield or shorten the breeding cycle.Also,the rubber genome editing systems developed in our present study provide practical ways for both functional analyses of endogenous genes and breeding of new varieties.The research results are as follows:1.Development of plasmid-based CRISPR/Cas9 rubber tree genome editing system:1.1 Vector constructionThe sequences of At U6-26 and Gh U6-9 were firstly used as the queries to blast rubber tree genome database(hevea.catas.cn),and 5 Hb U6 genes having over 99%identities with At U6-26 and Gh U6-9 were obtained.The promoter regions of these Hb U6 genes were analyzed,and respectively cloned into the genome editing vector carrying a Cas9 gene.Ten sg RNAs with high specificity were designed to target 2 FT(Hb FT1,Hb FT2)and 3 TFL1(Hb TFL1-1,Hb TFL1-2,Hb TFL1-3)genes,and constructed into the genome editing vectors,downstream of the U6 promoters,respectively.1.2 Efficiencies and patterns of targeted mutagenesisMutagenesis was successfully induced at the Hb TFL1-3 target site,with the sg RNA(T10)driven by the five distinct Hb U6 promoters,indicating that these five Hb U6 promoters can effectively drive the transcription of sg RNA.The mutagenesis efficiencies vary from 8.47% to 24,92% under different Hb U6 promoters.Among these,the promoters p Hb U6-2 and p Hb U6-4 show the highest editing efficiency,and the promoter p Hb U6-2 has the shortest sequence.Hence,the promoter p Hb U6-2 was selected as the specific promoter for subsequent experiments.Among the ten target sites,the highest editing efficiency was achieved at the target Hb TFL1-3(T10),with the sg RNAs driven by Hb U6-2 promoter.The target sequences were modified with+1 nt insertions or deletions.We also observed DNA fragment insertion at some target sites.2.CRISPR/Cas9 RNP rubber tree genome editing system:2.1 Editing SystemCas9 protein and in vitro transcribed sg RNA targeting Hb TFL1-3(T10)were assembled in different molar ratios and transformed into rubber tree protoplasts.Results showed that the molar ratio of Cas9/sg RNA at 1:7 result in considerable editing efficiency.Five specific sg RNAs targeting five rubber tree FT/TFL1 genes were in-vitro transcribed,pre-assembled with the Cas9 protein at a molar ratio of 1:7,and then transformed into rubber tree protoplasts.When multiple sg RNAs were transformed in one case,equal amounts of sg RNA transcript for each target were used and the total amount of sg RNAs remained the same as in single sg RNA samples.2.2 Efficiencies and patterns of targeted mutagenesisIn all single sg RNA transformed cases,targeted mutagenesis was successfully induced with frequencies ranging from 3.74% to 20.11%.Moreover,multiple mutations were successfully induced at the corresponding target sites using sg RNA combinations by a single transformation event,and the mutation frequencies at each target site varied from 1.26% to 10.32%,which were lower rates than that of the single sg RNA assay.Two mutation patterns including +1 nt insertions and deletions were detected at all target sites.We did not observe any DNA fragment insertion or nucleotide substitution at five target sites.Our study provides a promising first step to produce non-chimeric DNA-free genome-edited rubber tree plants in the future.