Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in <i>Arabidopsis thaliana</i>

Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5'-NG-3' PAM-recognizing variant of SpCas9 with adenosine deaminases TadA7.10 or TadA8e. Using a phenotype-based screen in <i>Arabidopsis thaliana</i> targeting multiple <i>PDS3</i> intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest adenine base editor (ABE) efficiency. With pECNUS4, we recreated a naturally occurring allele of <i>DANGEROUS MIX3</i> (<i>DM3</i>) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant <i>DM1/SSI4</i> homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T₂. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T₁ plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.