Base Editing in Poplar Through an Agrobacterium-Mediated Transformation Method

Abstract

CRISPR-Cas9 systems have revolutionized genome editing in plants and facilitated gene knockout and functional genomic studies in woody plants, like poplar. However, in tree species, previous studies have only focused on targeting indel mutations through CRISPR-based nonhomologous end joining (NHEJ) pathway. Cytosine base editors (CBEs) and adenine base editors (ABEs) enable C-to-T and A-to-G base changes, respectively. These base editors can introduce premature stop codons and amino acid changes, alter RNA splicing sites, and edit cis-regulatory elements of promoters. Base editing systems have only been recently established in trees. In this chapter, we describe a detailed, robust, and thoroughly tested protocol for preparing T-DNA vectors with two highly efficient CBEs, PmCDA1-BE3 and A3A/Y130F-BE3, and the highly efficient ABE8e as well as delivering the T-DNA through an improved protocol for Agrobacterium-mediated transformation in poplar. This chapter will provide promising application potential for precise base editing in poplar and other trees.