Progress and challenges in applying CRISPR/Cas techniques to the genome editing of trees

With the advent of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/CRISPR-associated protein(Cas)system,plant genome editing has entered a new era of robust and precise editing for any genes of interest.The development of various CRISPR/Cas toolkits has enabled new genome editing outcomes that not only target indel mutations but also enable base editing and prime editing.The application of the CRISPR/Cas toolkits has rapidly advanced breeding and crop improvement of economically important species.CRISPR/Cas toolkits have also been applied to a wide variety of tree species,including apple,bamboo,Cannabaceae,cassava,citrus,cacao tree,coffee tree,grapevine,kiwifruit,pear,pomegranate,poplar,ratanjoyt,and rubber tree.The application of editing to these species has resulted in significant discoveries related to critical genes associated with growth,secondary metabolism,and stress and disease resistance.However,most studies on tree species have involved only preliminary optimization of editing techniques,and a more in-depth study of editing techniques for CRISPR/Cas-based editing of tree species has the potential to rapidly accelerate tree breeding and trait improvements.Moreover,tree genome editing still relies mostly on Cas9-based indel mutation and Agrobacterium-mediated stable transformation.Transient transformation for transgene-free genome editing is preferred,but it typically has very low efficiency in tree species,substantially limiting its potential utility.In this work,we summarize the current status of tree genome editing practices using the CRISPR/Cas system and discuss limitations that impede the efficient application of CRISPR/Cas toolkits for tree genome editing,as well as future prospects.