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Abstract The canonical non-homologous end joining (c-NHEJ) repair pathway, generally viewed as stochastic, has recently been shown to produce predictable outcomes in CRISPR-Cas9 mutagenesis. This predictability, mainly in 1-bp insertions and small deletions, has led to the development of in-silico prediction programs for various animal species. However, the predictability of CRISPR-induced mutation profiles across species remained elusive. Comparing CRISPR-Cas9 repair outcomes between human and plant species reveals significant differences in 1-bp insertion profiles. The high predictability observed in human cells links to the template-dependent activity of human Polλ. Yet plant Polλ exhibits dual activities, generating 1-bp insertions through both templated and non-templated manners. Polλ knockout in plants leads to deletion-only mutations, while its overexpression enhances 1-bp insertion rates. Two conserved motifs are identified to modulate plant Polλ‘s dual activities. These findings unveil the mechanism behind species-specific CRISPR-Cas9-induced insertion profiles and offer strategies for predictable, precise genome editing through c-NHEJ. 

Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.