Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in Shewanella oneidensis MR-1

Abstract

Homologous recombination-mediated genomic editing is urgently needed to obtain high-performance chassis of electroactive microorganisms. However, the existing tools cannot meet the requirement of genome-wide editing in Shewanella oneidensis. Here, we develop different CRISPR-Cas systems that are ideal to be employed in AT-rich sequences as the supplements to Cas9. AsCpf1 and BhCas12b show low cell toxicity and superior ability to target sequences and are thus screened out in S. oneidensis MR-1. The PAMs of AsCpf1 and BhCas12b are 5′-TTTV-3′ and 5′-ATTN-3′. For gene deletion, ∼1-kb gene is knocked out and the editing efficiency is 41.67% by BhCas12b-mediated system. For gene replacement, endogenous promoter of nagK was substituted to a constitutive promoter with the efficiency of 25% through BhCas12b system. For gene insertion, the integration efficiency was up to 94.4% and 83.9% via CRISPR-BhCas12b and AsCpf1 tools. This study implies a great potential of CRISPR-BhCas12b/AsCpf1 systems recognizing AT-rich PAMs for genomic editing in S. oneidensis to facilitate multifaceted gene manipulation.

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

“…), which facilitates DNA fragment deletion and replacement, nucleotide changes, transcriptional repression and activation of target genes as well as artificial gene evolution [10,[21][22][23]. Alternatively, the type V CRISPR/Cas12a system, characterized by a single crRNA and a thymine-rich protospacer adjacent motif (PAM), is attractive and has been increasingly applied in bacterial genome editing in recent years [20,22,24]. In addition, the type I CRISPR systems, comprising the Cas3 nuclease and multiple effector proteins, have been reprogrammed to edit specific genome regions in its native hosts initially, and further successfully employed in heterologous editing in other bacterium species lacking the natural CRISPR systems [25][26][27].…”

“…Our current knowledge about CRISPR-mediated genome editing in bacteria mainly comes from some human pathogens as well as industrial and laboratory strains, such as Escherichia coli [ 9 – 11 ], Pseudomonas [ 12 , 13 ], Mycobacterium [ 14 ], Bacillus [ 15 , 16 ], Corynebacterium [ 17 ], Clostridium [ 18 ], Streptomyces [ 19 ], etc . Generally speaking, the type II CRISPR/Cas9 system [ 20 ] is the most widely-used genome editor in various bacterial strains due to its robust nuclease activity and broad compatibility with other functional proteins (i.e. nucleotide deaminases, transcriptional regulators, DNA polymerase, reverse transcriptase, etc .…”

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 ].…”

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

“…Our current knowledge about CRISPR-mediated genome editing in bacteria mainly come from a number of human pathogens as well as industrial and laboratory strains, such as Escherichia coli [8–10], Pseudomonas [11, 12], Mycobacterium [13], Bacillus [14, 15], Corynebacterium [16], Clostridium [17], Streptomyces [18], etc . Generally speaking, the type II CRISPR/Cas9 system[19] is the most widely-used heterologous genome editor in various bacterial strains due to its robust nuclease activity and broad compatibility with other functional proteins (i.e. nucleotide deaminases, transcriptional regulators, DNA polymerase, reverse transcriptase etc .…”

“…), which facilitates DNA fragment deletion and replacement, nucleotide changes, transcriptional depression and activation of target genes as well as artificial gene evolution [9, 20–22]. By contrast, the type V CRISPR/Cas12a system, characterized by a single crRNA and a thymine-rich protospacer adjacent motif (PAM), has been attractive and increasingly applied in bacterial genome editing in recent years [19, 21, 23]. Alternatively, the endogenous type I CRISPR systems, comprising the Cas3 nuclease and multiple effector proteins, have also been successfully reprogrammed to self-edit specific genome regions in its native hosts [24–26].…”