In Vivo Genome Editing in Type I and II Methanotrophs Using a CRISPR/Cas9 System

Abstract

Methanotrophic bacteria are Gram-negative, aerobic organisms that use methane as their sole source of carbon and energy. In this study, we constructed and exemplified a CRISPR/Cas9 genome editing system and used it to successfully make gene deletions and insertions in the type I methanotroph Methylococcus capsulatus Bath and the type II methanotroph Methylocystis parvus OBBP. High frequencies of gene deletions and insertions were achieved in combination with homology-directed repair. In M. parvus OBBP, we also investigated the impact of several parameters on the CRISPR/Cas9 genome editing, where the ligD gene was targeted with various PAM sequences and guide RNA spacer sequences, homology arms of variable length, differences in the duration of mating during conjugation, and exploiting promoters of different strengths to control the expression of cas9 and sgRNA. Although not the first attempt to develop a CRISPR/Cas system in methanotrophs, this work demonstrated for the first time an efficient CRISPR/Cas9 system generating scarless clean gene deletions and insertions in methanotroph genomes.

Construction of a broad-host-range Anderson promoter series and particulate methane monooxygenase promoter variants expand the methanotroph genetic toolbox

“…Several inducible promoter systems reliant on allosterically regulated transcriptional regulators (e.g. TetR, AraC) have also been shown to function in industrially relevant methanotrophs [ [15] , [16] , [17] ], which have enabled advanced methanotroph gene editing technologies like CRISPR-Cas to be developed [ 10 , 18 , 19 ]. Collectively, BHR plasmids and regulatory genetic parts have been used to express native, heterologous, and synthetic biochemical pathways in engineered methanotrophs to produce valuable molecules directly from CH 4 [ 6 , 20 ] and references therein).…”

Modular and Flexible Molecular Device for Simultaneous Cytosine and Adenine Base Editing at Random Genomic Loci in Filamentous Fungi

“…Recent developments in synthetic biology, metabolic engineering, multiomics, high-throughput DNA sequencing and synthesis, and computational biology have led to a growing need for efficient and reliable genetic engineering techniques to accelerate strain development in a design-build-test-learn cycle . The rational engineering of genomic alterations via genomic editing technologies represents a substantial breakthrough in molecular biology, with broad applications in various biotechnological fields . Currently, the most commonly used genome editing techniques include base editing, CRISPR-Cas9, TALENs, ZFNs, and prime editing. , …”

“…1 The rational engineering of genomic alterations via genomic editing technologies represents a substantial breakthrough in molecular biology, with broad applications in various biotechnological fields. 2 Currently, the most commonly used genome editing techniques include base editing, CRISPR-Cas9, TALENs, ZFNs, and prime editing. 3,4 Base editing, an accurate and effective method, enables the direct conversion of one specific base to another, circumventing the requirement for double-stranded breaks in the DNA, via the utilization of various base editors (BEs), such as cytosine base editors (CBEs) and adenine base editors (ABEs).…”