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.

Keywords: CRISPR; DNA ligase; gene deletion; gene insertion; genome editing; homology-directed repair; methane monooxygenase; methanotrophs; promoter library.

Figure 1

Fluorescence assay of promoters. (A) pMTL94115_P plasmid used for promoter assay. Fluorescence assay of promoters in (B) E. coli S17-1 λpir; (C) M. parvus OBBP; (D) M. capsulatus Bath. All promoters are expressed constitutively using eYFP as the reporter. Fluorescence values for promoterless eYFP was used to normalize all promoter-eYFP data.

Figure 2

Plasmids, CE, and genome editing screens of M. parvus OBBP. (A) Plasmid pMTL9BR2-Cas9 carrying S. pyogenescas9 gene cloned into pMTL94111 backbone and subsequent plasmids designed from pMTL9BR2-Cas9. (B) CE graph showing significant difference between pMTL9BR2-Cas9 when compared to pMTL9BR2-Cas9-gRNA_phaC or pMTL9BR2-Cas9_ΔligD (P = 0.0088). pMTL9BR2-Cas9-gRNA_ligD CE was significantly lower than pMTL9BR2-Cas9_ΔligD (P = 0.02). There was no significant difference between pMTL94111 and pMTL9BR2-Cas9 (P = 0.3681). Unpaired t-test used, n = 2, error bars represent the standard error of the mean. (C) Bands of PCR screens of unsuccessful phaC gene deletion. phaC is a 2070 bp gene, and edited colonies were expected to have bands approximately 2070 bp smaller than the control (WT), which is in the last lane. (D) Bands of PCR screen of successful ligD gene deletion. ligD is 2448 bp in size, and edited colonies were expected to have bands approximately 2448 bp smaller than the control (M. parvus OBBP WT gDNA), which is in the last lane. The numbers of colonies screened were represented by numbers. NEB 1 kb Plus DNA ladder was used. Dotted lines represent the size of WT PCR amplicon, while solid lines represent mutants.

Figure 3

ΔligD gene deletion under different conditions (A) CE of additional ligD deletions in M. parvus OBBP. (B) M. parvus OBBP ligD gene deletion using pMTL9BR2-Cas9_ΔligD24 plasmid conjugated with E. coli (24 h mating time). (C) M. parvus OBBP ligD gene deletion using pMTL9BR2-Cas9_ΔligD48 plasmid conjugated with E. coli (48 h mating time). (D) M. parvus OBBP ligD gene deletion using pMTL9BR2-Cas9Pals_ΔligD plasmid with a relatively stronger Cas9 promoter (P_als) compared to sgRNA promoter (P_mdh) conjugated with E. coli. (E) M. parvus OBBP ligD gene deletion using pMTL9BR2-Cas9-ΔligD_HA500 plasmid with 500 bp HA conjugated with E. coli. NEB 1 kb Plus DNA ladder was used. Dotted lines represent the size of WT PCR amplicon, while solid lines represent mutants. The number of colonies screened were represented by numbers.

Figure 4

Deletion of additional genes in M. parvus OBBP. (A) M. parvus OBBP gene deletion using pMTL9BR2-Cas9_ΔpntA plasmid. (B) M. parvus OBBP gene deletion using pMTL9BR2-Cas9_ΔbcsB plasmid. (C) M. parvus OBBP gene deletion using pMTL9BR2-Cas9_ΔMPA_0518 plasmid. NEB 1 kb Plus DNA ladder was used. Dotted lines represent size of WT PCR amplicon, while solid lines represent mutants. Lanes 1–10 represent the 10 colonies screened.

Figure 5

Gene insertion in M. parvus OBBP. (A) plasmid pMTL9BR2-Cas9-eYFP_KI1 used for inserting eYFP gene and simultaneous replacement of ligD gene. (B) Plasmid pMTL9BR2-Cas9-eYFP_KI2 used for insertion of eYFP gene without gene replacement. (C) eYFP and its P3 promoter are both 1091 bp long. Using pMTL9BR2-Cas9-eYFP_KI1 to Replace ligD with eYFP will give a band around 1357 bp smaller than the control which is M. parvus OBBP WT gDNA. (D) pMTL9BR2-Cas9-eYFP_KI2 colonies with successful gene insertions have bands approximately 1091 bp higher than the control, which is M. parvus OBBP WT gDNA. The same primer set was used for screening in (C,D). NEB 1 kb Plus DNA ladder was used. Dotted lines represent size of WT PCR amplicon, while solid lines represent mutants. Lanes 1–10 represent the 10 colonies screened.

Figure 6

Genome editing in M. capsulatus Bath. (A) M. capsulatus Bath mmoX gene deletion plasmid pMTL9BR1-Cas9_ΔmmoX. (B) M. capsulatus Bath mmoX gene deletion PCR screen. (C) M. capsulatus Bath gene deletion using pMTL9BR1-Cas9_ΔczcA plasmid. (D) M. capsulatus Bath gene deletion using pMTL9BR1-Cas9_ΔMCA_0145 plasmid. (E) M. capsulatus Bath gene deletion using pMTL9BR1-Cas9_ΔMCA_2158 plasmid. (F) Gene insertion of eYFP with simultaneous gene deletion of MCA_0145 using plasmid pMTL9BR1-Cas9_ΔMceYFPKI1. NEB 1 kb Plus DNA ladder was used. Dotted lines represent the size of WT PCR amplicon, while solid lines represent mutants.