Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase

Abstract

Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN, which codes for nattokinase in Bacillus subtilis, was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC Finally, the engineered strain DWc9nΔ7 (Δepr ΔwprA Δmpr ΔaprE Δvpr ΔbprA ΔbacABC), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis This tool could be applied to strain improvement for future research.IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

Keywords: Bacillus licheniformis; CRISPR-Cas9n; deletion; genome editing; integration; nattokinase production.

FIG 1

Expression and integration of Cas9 nickase in B. licheniformis. (A) SDS-PAGE analysis of the intracellular proteins in DW2/pHY300 and DW2/pcas9n. Lane M, 200-kDa protein marker (200, 150, 120, 100, 85, 70, 60, and 50 kDa); lane 2, DW2/pHY300; lane 3, DW2/pcas9n. The arrow indicates the Cas9 nickase. (B) Confirmation of Cas9n integration strain by PCR amplification. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 1, PCR product of DW2 using Cas9n-F and Cas9n-R (no DNA bond with this control strain); lane 2, PCR product of DWc9n using Cas9n-F and Cas9n-R (4,933 bp). (C) The construction procedure for the Cas9n integration expression strain DWc9n.

FIG 2

Precise deletion of single gene and efficiency evaluation. (A) The strategy for knocking out yvmC in B. licheniformis by CRISPR-Cas9n system. (B) Confirmation of the yvmC-deficient strain by PCR amplification. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 1, PCR product of DWc9n using Y-F/Y-R (3,122 bp); lane 2, PCR product of DWc9n using M-F/Y-R (1,792 bp); lane 3, PCR product of DWc9n△yvmC using Y-F/Y-R (2,427 bp); lane 4, PCR product of DWc9n△yvmC using M-F/Y-R (no DNA bond for lacking the target sequence of the primer M-F). (C) Effects of different sizes of homologous arms on the efficiency of yvmC deletion.

FIG 3

Precise disruption of epr and wprA simultaneously. (A) Plasmid construction procedure for double gene (epr and wprA) deletion. (B) Confirmation of epr- and wprA-deficient strain by PCR amplification using primers △epr-Y-F/Y-R and △wprA-Y-F/Y-R. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 2, PCR product of DWc9n using primers △epr-Y-F/Y-R (2,328 bp); lane 3, PCR product of DWc9n△epr△wprA using primers △epr-Y-F/Y-R (1,428 bp); lane 4, PCR product of DWc9n using primers △wprA-Y-F/Y-R (3,802 bp); lane 5, PCR product of DWc9n△epr△wprA using primers △wprA-Y-F/Y-R (2,805 bp).

FIG 4

Precise deletion of a large DNA fragment. (A) The construction procedure for the bacitracin synthase gene cluster bacABC-deficient strain. (B) Confirmation of bacABC-deficient strain by PCR amplification using primers △bacABC-Y-F/Y-R. Lane M, DL 5000 marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 1, PCR product of bacABC in DWc9n (too large to amplify); lane 2, PCR product of DWc9n△bacABC (1,977 bp). The verified primers are designed at both ends of bacABC (Y-F and Y-R) and the PCR product size is 1,977 bp in the bacABC-deficient strain, whereas no PCR product was found for the wild-type strain on gel electrophoresis. (C) Confirmation of bacABC-deficient strain by DNA sequence.

FIG 5

Precise insertion of aprN expression cassette. (A) The construction procedure for aprN integration strain. (B) Confirmation of aprN integration by PCR amplification using primers NK-Y-F/Y-R. Lane M, DL 5000 marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 2, PCR product of DWc9n using primers NK-Y-F/Y-R (1,592 bp); lane 3, PCR product of aprN integration strain using primers NK-Y-F/Y-R (2,927 bp). The star represents the aprN integration strain. (C) Sequencing analysis of integrated site in DWc9n.

FIG 6

Confirmation of the extracellular protease gene-deficient strain by PCR amplification. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 2, PCR product of DWc9n using primers △epr-Y-F/Y-R (2,328 bp); lane 3, PCR product of DWc9n△7 using primers △epr-Y-F/Y-R (1,428 bp); lane 4, PCR product of DWc9n using primers △wprA-Y-F/Y-R (3,802 bp); lane 5, PCR product of DWc9n△7 using primers △wprA-Y-F/Y-R (2,805 bp); lane 6, PCR product of DWc9n using primers △mpr-Y-F/Y-R (2,054 bp); lane 7, PCR product of DWc9n△7 using primers △mpr-Y-F/Y-R (1,112 bp); lane 8, PCR product of DWc9n using primers △aprE-Y-F/Y-R (1,791 bp); lane 9, PCR product of DWc9n△7 using primers △aprE-Y-F/Y-R (945 bp); lane 10, PCR product of DWc9n using primers △vpr-Y-F/Y-R (1,882 bp); lane 11, PCR product of DWc9n△7 using primers △vpr-Y-F/Y-R (886 bp); lane 12, PCR product of DWc9n using primers △bprA-Y-F/Y-R (2,258 bp); lane 13, PCR product of DWc9n△7 using primers △bprA-Y-F/Y-R (742 bp); lane 14, PCR product of DWc9n using primers △bacABC-Y-F/Y-R (too large to amplify); lane 15, PCR product of DWc9n△7 using primers △bacABC-Y-F/Y-R (1,977 bp).

FIG 7

Time profiles of nattokinase production and cell growth in DWc9n/pP43SNT-SsacC and DWc9n△7/pP43SNT-SsacC strains.