Repurposing CRISPR RNA-guided integrases system for one-step, efficient genomic integration of ultra-long DNA sequences

Abstract

Genomic integration techniques offer opportunities for generation of engineered microorganisms with improved or even entirely new functions but are currently limited by inability for efficient insertion of long genetic payloads due to multiplexing. Herein, using Shewanella oneidensis MR-1 as a model, we developed an optimized CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST system), which enables programmable, RNA-guided transposition of ultra-long DNA sequences (30 kb) onto bacterial chromosomes at ∼100% efficiency in a single orientation. In this system, a crRNA (CRISPR RNA) was used to target multicopy loci like insertion-sequence elements or combining I-SceI endonuclease, thereby allowing efficient single-step multiplexed or iterative DNA insertions. The engineered strain exhibited drastically improved substrate diversity and extracellular electron transfer ability, verifying the success of this system. Our work greatly expands the application range and flexibility of genetic engineering techniques and may be readily extended to other bacteria for better controlling various microbial processes.

Functional characterization of diverse type I-F CRISPR-associated transposons

“…Given the variation in integration distance, these genome editing tools cannot currently be used for integration events requiring precise, nucleotide-level insertions at a fixed distance from the target site. Still, many genome engineering goals, especially those incorporating large DNA insertions, are not hindered by such limitations, and a recent study applied ShCAST to ultra-long DNA insertions for metabolic engineering ( 45 ). We note that our PCR-based detection method may have failed to capture distant integration events, and further studies are required to elucidate the mechanisms behind integration distance variation with regards to system, target site, transposon size, TnsB binding site identity and configuration, and biological context.…”

Engineering extracellular electron transfer pathways of electroactive microorganisms by synthetic biology for energy and chemicals production

“…Interestingly, the ShCAST system could be rapidly eliminated by arabinose-induced expression of I-SecI endonuclease, thus enabling the rapid iterative integration of genes. 110…”

“…Interestingly, the ShCAST system could be rapidly eliminated by arabinose-induced expression of I-SecI endonuclease, thus enabling the rapid iterative integration of genes. 110 2.2.3.2 Single base-editing systems. The dsDNA breaks (DSBs) generated by the CRISPR/Cas systems need to be repaired by homologous recombination (HR) with the template DNA or nonhomologous end joining (NHEJ) in eukaryotes to introduce specific mutations.…”

Highly efficient multiplex base editing: One-shot deactivation of eight genes in Shewanella oneidensis MR-1

“…Gene regulation tools include CRISPR interference (CRISPRi) for gene downregulation [ 9 ], CRISPR activation (CRISPRa) for gene upregulation [ 10 ], and CRISPR-PAIR for multi-mode regulation [ 11 ]. Meanwhile, gene editing tools have realized plenty of functions such as gene deactivation [ 12 ], gene knockout [ 13 ], gene replacement [ 14 ], gene insertion [ 15 ], and insertion of large fragments [ 16 ]. Amidst these technologies, base editing has been generally acknowledged as an efficacious way to deactivate genes, circumventing user-unfriendly gene knockout with introduction of DNA double-strand breaks and multiple components (e.g., ssDNA repair template) [ 17 ].…”