Cpf1(Cas12a)-based genome editing in the filamentous cyanobacterium Nostoc punctiforme

A Novel, Simple, and Highly Efficient Dual-sgRNA CRISPR/Cas9-Based Editing System in the Filamentous Fungus Fusarium oxysporum

A novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower.

CRISPR/Cas9 gene editing in the heterotrophic protist Acanthamoeba castellanii

Homologous knock-in processes facilitated by CRISPR-Cas9 in Aurantiochytrium and disrupting the gene for DNA ligase IV prevents transformation events

The potential of these CRISPR-Cas9 systems to serve as a robust foundation for the functional genomics and metabolic engineering of A. limacinum is demonstrated.

Cas3-mediated genome reduction: demonstration in Cupriavidus necator H16 improves growth on heterotrophic and autotrophic carbon sources

Abstract Genome reduction is widely used to improve microbial bioprocessing hosts by reducing the burden of inessential physiology. Rationally identifying genomic regions that are dispensable or even detrimental to bioprocessing is challenged by our inability to map genome sequence to function across complex regulation and physiology. Thus, there is a need for tools that rapidly generate reduced genome strains with improved performance in process-relevant conditions. Here, we report a Cascade–Cas3-enabled method called TRIM3 that generates large deletions by targeting a randomly integrated transposon, enabling facile generation of a genome-reduced mutant library. Mutants with improved performance were isolated following growth-coupled selection and analyzed by long-read DNA sequencing to identify deletions in their genomes. We deploy this system iteratively in the industrial host Cupriavidus necator H16 on fructose and on formate. After two rounds of TRIM3, we isolate a strain containing a total reduction of 1.4 Mb (18.4% of the genome) that grows 25% faster in a bioreactor on fructose and a strain with a total reduction of 0.5 Mb (7.3% of the genome) that grows 14% faster on formate. This work demonstrates a method for random, iterative, growth-selectable genome reduction that represents a new avenue for large-scale genome modifications and the development of improved bioprocessing hosts.

Establishment of an efficient and PAM-relaxed LbCas12a genome editing tool in plants.

This study expands Cas12a targeting scope in plants and provides the first evidence for Cas12-mediated MMEJ-based large-fragment deletion in plants.

Establishment of an Efficient CRISPR-Cas9-Mediated Gene Disruption System in the Lichen-Forming Fungus Umbilicaria muhlenbergii

Compared with conventional homologous recombination, the CRISPR-Cas9 system substantially improved gene disruption efficiency, thereby overcoming a major limitation in the genetic manipulation of lichen-forming fungi.