CRISPR-Cas12a-Assisted Genome Editing in Amycolatopsis mediterranei

Abstract

Amycolatopsis mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

“…The resulting clones were called A. mediterranei DSM 40773-pRIF12-pRIF14 and were used in further steps and for phenotypic analysis. From the literature, it appears that the efficiency of homologous recombination in Amycolatopsis spp is strain dependent (2, 13). Thus it might be preferable to use rather large recombination regions to integrate the attL and attR sequences.…”

“…Recent attempts to use CRISPR-Cas9 based systems in Amycolatopsis reveal a toxicity effect of Cas9 expression. A genome editing system based on other Cas protein was developed to obtain deletion mutants in Amycolatopsis mediterranei U32 (13), however this implicated the integration of the Cas12a gene and of the hygromycin resistance gene in the chromosome of A. mediterranei U32. A more common technique for gene inactivation is the replacement or deletion of the target gene by homologous recombination, the efficiency of which varies greatly depending on the strain (2), imposing the use of antibiotic resistance marker to select knockout mutants (14).…”

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

“…Among them, homology-directed repair (HDR) is the predominant mechanism for DSB repair in bacteria, which utilizes a homologous template and multi-subunit helicase/nuclease complexes to drive homozygous recombination between the damaged DNA strand and donor, leading to the accurate repair of DSBs [28]. Non-homologous end joining (NHEJ) is a template-independent DSB repair pathway available in only 20-25% of bacteria including Bacillus subtilis [28], M. tuberculosis [14], Pectobacterium atrosepticum [29], Amycolatopsis mediterranei [30], etc. Only two proteins, such as the DNA-end-binding protein Ku and a multifunctional ATP-dependent ligase (LigD), are required in the simplified NHEJ machine.…”

“…Non-homologous end joining (NHEJ) is a template-independent DSB repair pathway available in only 20–25% of bacteria including Bacillus subtilis [ 28 ], M . tuberculosis [ 14 ], Pectobacterium atrosepticum [ 29 ], Amycolatopsis mediterranei [ 30 ], etc . Only two proteins, such as the DNA-end-binding protein Ku and a multifunctional ATP-dependent ligase (LigD), are required in the simplified NHEJ machine.…”

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

“…As reported in prokaryotes so far, double-strand DNA breaks (DSBs) in genomic DNA could be repaired through three different mechanisms: homology-directed repair (HDR), classical non-homologous end joining (NHEJ) and alternative end joining (AEJ; also referred to as microhomology-mediated repair, MMEJ), the latter two mechanisms are independent of DNA repair template [27][28][29]. However, most bacteria do not have NHEJ/AEJ pathways with a few exceptions, such as E. coli [28], P. aeruginosa [30], B. subtilis [27], M. tuberculosis [13], R. capsulatus [31], A. mediterranei [32], etc. Thus, DSB introduced by Cas nucleases in the bacterial chromosome is severely toxic to cells and HDR-mediated genome editing with exogenous donor have been widely utilized in bacteria.…”