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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.

Prime editors are reverse transcriptase (RT)-based genome-editing tools that utilize double-strand break (DSB)-free mechanisms to decrease off-target editing in genomes and enhance the efficiency of targeted insertions. The multiple prime editors that have been developed within a short span of time are a testament to the potential of this technique for targeted insertions. This is mainly because of the possibility of generation of all types of mutations including deletions, insertions, transitions, and transversions. Prime editing reverses several bottlenecks of gene editing technologies that limit the biotechnological applicability to produce designer crops. This review evaluates the status and evolution of the prime editing technique in terms of the types of editors available up to prime editor 5 and twin prime editors, and considers the developments in plants in a systematic manner. The various factors affecting prime editing efficiency in plants are discussed in detail, including the effects of temperature, the prime editing guide (peg)RNA, and RT template amongst others. We discuss the current obstructions, key challenges, and available resolutions associated with the technique, and consider future directions and further improvements that are feasible to elevate the efficiency in plants.

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5′ overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5′ overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.

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.

The advancement in high power lasers has urged the requisite of efficient optical limiting materials for both eye and sensor protection. The discovery of atomically thin 2D transition metal dichacogenides with distinctive properties has paved the way for a variety of applications including optical limiting. Until recently, the optical limiting effect exhibited by 2D materials is inferior to the benchmark materials fullerene (C₆₀) and graphene. This article reports the optical limiting activity of the 2D transition metal dichalcogenide, titanium disulfide (TiS₂) nanosheets, using optical and photoacoustic z‐scan techniques. The 77% nonlinear optical limiting exhibited by the TiS₂sheets with 73% linear‐transmittance is superior to that of any other existing 2D dichalcogenide sheets, graphene, and the benchmark optical limiting material, C₆₀. The enhanced nonlinear response is attributed to the concerted effect of 2‐photon and the induced excited state absorptions. By using photoacoustic z‐scan, a unique tool developed to determine the nonlinear optical limiting mechanism in materials, it is found that the optical limiting exhibited by TiS₂2D sheets and graphene are mainly due to nonlinear absorption rather than scattering effects. These results have opened the door for 2D‐dichalcogenide‐materials‐based highly efficient optical limiters, especially at low fluences.