Cas12a-guided platform for precise multi-kilobase DNA insertion in dicot crops

Overview

Nulla Bio offers a Cas12a-guided targeted-insertion platform designed to overcome the three linked bottlenecks that limit large-fragment DNA integration in dicot crops: insufficient donor availability, low productive donor use at the break site, and competing non-homologous end joining (NHEJ). The platform delivers precise, homology-directed repair (HDR)-resolved insertion of 7–10 kb payloads at predefined genomic loci, with a roadmap toward larger cargos and multi-locus stacking. It uses LbCas12a exclusively and operates within standard 25–30 °C regeneration windows, avoiding heat-shock steps. Phase 1 is anchored in tomato, with soybean and oilseed rape following in subsequent phases.

Technical specifications

Core technology layers:

• Geminiviral replicon donor amplification raises donor copy number at the target site, leveraging bean yellow dwarf virus (BeYDV)-based replicon delivery previously shown to increase somatic gene targeting roughly three-fold with LbCas12a outperforming SpCas9.
• KUDN-tethered Cas12a (a dominant-negative KU80 fusion) biases repair away from canonical NHEJ, previously demonstrated to improve gene targeting 1.7–3.55-fold across loci and up to 9.8-fold at a specific locus.
• SunTag-assembled ORF1 donor recruitment concentrates donor DNA at the double-strand break, raising local donor availability at the editing site.
• Chemical-inducible GR/Dex layer synchronizes donor build-up, donor release, and target cleavage within a single productive window, with a beta-estradiol/XVE variant available as backup induction.

Benchmarking and optimization variables:

• Three configurations evaluated under matched conditions: SunTag-assembled ORF1 recruitment, a KUDN-Cas12a-ORF1-GR mega-fusion, and an external Cas12a-based comparator retained for benchmarking.
• Tunable parameters include donor architecture, SunTag repeat number (5x/10x/24x), anchor-to-payload stoichiometry, dexamethasone dose, and induction timing and washout at 28–30 °C.
• Event quality assessed by junction sequencing, copy-number analysis, backbone exclusion, and long-read confirmation.

Intellectual property: Three patent families (KR/US/CN/CA) protect the core platform components.

Technology readiness level

The platform builds on individually validated backbone layers from prior peer-reviewed work in tomato. Phase 1 (Year 1) benchmarks the three configurations in tomato against a defined go/no-go criterion of 2.5% precise single-copy insertion of a 7–10 kb payload. Achievement of this milestone triggers Phase 2 (Year 2), extending the platform to soybean with adapted induction dosing for cotyledonary-node explants and to oilseed rape via the R2S-T regeneration route validated in Brassica oleracea. A conditional Phase 3 addresses scaling toward 25 kb payloads and multi-locus stacking in allotetraploid Brassica napus. The technology is currently at a validated-component stage, with integrated platform performance being actively demonstrated.