CRISPR/Cas12a toolbox for genome editing in Methanosarcina acetivorans

Abstract

Methanogenic archaea play an important role in the global carbon cycle and may serve as host organisms for the biotechnological production of fuels and chemicals from CO₂ and other one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to its large genome, versatile substrate range, and available genetic tools. Genome editing in M. acetivorans via CRISPR/Cas9 has also been demonstrated. Here, we describe a user-friendly CRISPR/Cas12a toolbox that recognizes T-rich (5'-TTTV) PAM sequences. The toolbox can manage deletions of 3,500 bp (i.e., knocking out the entire frhADGB operon) and heterologous gene insertions with positive rates of over 80%. Cas12a-mediated multiplex genome editing was used to edit two separate sites on the chromosome in one round of editing. Double deletions of 100 bp were achieved, with 8/8 of transformants being edited correctly. Simultaneous deletion of 100 bp at one site and replacement of 100 bp with the 2,400 bp uidA expression cassette at a separate site yielded 5/6 correctly edited transformants. Our CRISPR/Cas12a toolbox enables reliable genome editing, and it can be used in parallel with the previously reported Cas9-based system for the genetic engineering of the Methanosarcina species.

Keywords: CRISPR/Cas12a; Methanosarcina acetivorans; genome editing; methanogens; synthetic biology.

Figure 1

Construction of the Cas12a-gRNA expression system for M. acetivorans. (A) Schematic diagram of pMCp4 and Cas12a-gRNA expression system. The plasmid pMCp4 expresses the Cas12a protein in M. acetivorans. The Cas12a-gRNA expression system expresses the Cas12a-gRNA complex in M. acetivorans. Cas12a and gRNA cassettes are equipped with tetracycline-regulated promoter PmcrB (tetO1) and the promoter PmtaC1 from M. acetivorans, respectively. (B) Targeting efficiency of the Cas12a-gRNA expression system. Cas12a-only, plasmid pMCp4. g1RNA, g2RNA, g3RNA, g4RNA, and g5RNA, five gRNAs designed for targeting various locations along the ssuC gene. Error bar represents the standard deviation of triplicate measurements. Standard deviations were not determined for gRNA-expressing transformation data, as all cells were plated out to analyze the lethal efficiency of the Cas12a-gRNA complex.

Figure 2

CRISPR/Cas12a-mediated gene knockout. (A) Schematic diagram of Cas12a-gRNA gene editing system. Cas12a and gRNA cassettes are equipped with promoters PmcrB (tetO1) and PmtaC1, respectively. Gene editing was achieved by the introduced upstream and downstream homologous repair (HR) arms. (B) Scheme for generating gene deletions in ssuC. g1RNA was designed to target ssuC to form a double-stranded break (DSB). Various sizes of gene knockouts were generated by introducing a 1,000-bp length of flanking HR sequence near the leakage site. Δ100 bp, Δ500 bp, Δ1000 bp, and Δ2000 bp, plasmids generating 100 bp, 500 bp, 1,000 bp, and 2000 bp of gene knockouts while repairing the DSB. (C) Transformation efficiency of deletion-generating plasmids. Cas12a-g1RNA, plasmid expressing Cas12a-g1RNA complex that targets ssuC to produce the DSB on the genome. Cas12a-only, plasmid pMCp4 expresses Cas12a. Error bars represent the standard deviation of triplicate measurements. Standard deviations were not determined for Cas12a-g1RNA transformation data, as all cells were plated out to analyze the lethal efficiency of the Cas12a-g1RNA complex. (D) Editing efficiency of deletion-generating plasmids. Ten Pur^R transformants were randomly selected for colony PCR. WT, wild type M. acetivorans strain. Thermo Scientific™ GeneRuler 1 kb DNA ladder was used for sizing DNA fragments.

Figure 3

CRISPR/Cas12a-mediated gene insertion. (A) Transformation efficiency of Cas12a-mediated gene insertion. Cas12a-g1RNA, plasmid pMCp2-g1RNA expressing Cas12a-g1RNA complex that targets ssuC to produce the DSB on the genome. Cas12a-only, plasmid pMCp4 expresses Cas12a. Δ100 bp::uidA, plasmid pMCp3-g1-100-uid replaced 100 bp with the uidA expressing cassette in genome. Error bars represent the standard deviation of triplicate measurements. Standard deviations were not determined for Cas12a-g1RNA transformation data, as all cells were plated out to analyze the lethal efficiency of the Cas12a-g1RNA complex. (B) Editing efficiency of gene insertion-generating plasmid. Upper panel, scheme for the engineered genome containing uidA cassette and the detecting primers used in colony PCR. veri8 and veri2, forward and reverse primer target uidA and genome. HR-upstream-1 and HR-downstream-1, the flanking HR sequence identical to the ones used in Figure 2B. Bottom panel, 20 Pur^R transformants were randomly selected for colony PCR to verify the existance of the uidA gene. Plas. and WT are plasmid pMCp3-g1-100-uid and wild type M. acetivorans genome, respectively, and served as negative controls. Thermo Scientific™ GeneRuler DNA Ladder Mix was used for sizing DNA fragments.

Figure 4

CRISPR/Cas12a-mediated multiplex genome editing. (A) The CRISPR array designed for targeting two sites on the genome. g1RNA and g9RNA target ssuC and frhA, generating two DSBs in the genome simultaneously. DR, direct repeat sequences in the gRNA cassette. (B) Editing efficiency of the multiplex gene knockout. Upper panel, scheme for the engineered genome with 100 bp-deletions in both ssuC and frhA sites and the primers used in colony PCR. veri1 and veri2, forward and reverse primer target HR sequence and the downstream of ssuC. veri11 and Cp54, forward and reverse primer target the upstream and the downstream of frhA. Bottom panel, positive rate of multiplex gene knockout. Eight transformants were randomly selected for colony PCR to verify the gene knockout efficiency. Plas. and WT are plasmid pMCp3-g1g9–100 and wild type M. acetivorans strain, respectively, and served as the negative controls. g1Δ100, gene deletion in ssuC. g9Δ100, gene deletion in frhA. Thermo Scientific™ GeneRuler 1 kb DNA ladder was used for sizing DNA fragments.