Construction of CRISPR vector of Eucalyptus grandis CTX gene family by pHDE-Cas9

ObjectiveInterspaced short palindromic repeats(CRISPR/Cas)vectors of Eucalyptus grandis whose cy-tokinin oxidase/dehydrogenase(CTX)gene family was regularly clustered were constructed by efficient gene editing sys-tem(pHDE-Cas9)to lay a foundation for verification of the functions of E.grandis CTX gene family.MethodTarget sites and primers of gene CTX in E. grandis family were designed,target fragment on pCBC plasmid was amplified, pHDE vector was digested with Bsa I,after that they were ligated into target fragment to form recombinant plasmids.The recombinant plasmids were transformed into DH5α competent cells.The monoclonal colonies were picked up for prelimi-nary confirmation by the bacterial liquid PCR,recombinant plasmid PCR verification,and then the positive cloned plas-mids were sequenced,and CRISPR vector of CTX gene family was constructed.ResultThe recombinat plasmids PCR identification and sequencing indicated that,there was no mutation in recombinat plasmid sequence,and it was highly consistent with the expected sequence.Homologous expression vectors pHDE-CTXA,pHDE-CTXB and pHDE-CTXC of CTX family in E.grandis were constructed.ConclusionBy using pHDE-Cas9,point editing vector of CTX gene family in E.grandis is constructed,and the procedure is rapid and efficient.It establishes a foundation for future studies in ge-nome point editing vector of eucalyptus and construction of other eucalyptus CRISPR/Cas9 expression vectors.