Table 2 Strains and plasmids used in this study

Strains

 E. coli DH5α

F-φ80lacZΔM15Δ(lacZYA-argF)Μ169enA1recA1hsdR17(rk-,mk-)sμpE44λ-thi-1gyrA96relA1phoA

WeiDi (Shang hai)

 B. subtilis SCK6

Δhis, ΔnprRn, ΔnprE18, lacA::P_xylA-comK, aprA3 eglS102, bglT, bglSRV, Erm^R

Laboratory preservation

 BS01

B. sublitis SCK6 derivative with its amyE deleted

This study

 BS02

B. sublitis SCK6 derivative with its spo0A deleted

This study

 BS03

BS01 derivative with epr and bpr deleted

This study

 BS04

B. subtilis SCK6 + pHY-SDFE27

This study

 BS05

BS02 + pHY-SDFE27

This study

 BS06

BS03 + pHY-SDFE27

This study

Plasmids

 pHY300PLK

E. coli-B. subtilis shutter plasmid, Tet^R, Amp^R

Laboratory preservation

 pCRISPomyces-2

Streptomyces expression of codon-optimized Cas9 and custom gRNA

addgene#61737

 pHY-ngCas9

pHY300PLK derivative, P_glyA-BsaI-spacer-BsaI-gRNAscffold-T1 terminator, P_acoR-gRNA_rep, P_liaI-Cas9

This study

 pGE-KOerm

pHY-ngCas9 derivative, 20 bp gRNA targeting erm, 1000 bp donor DNA(deletion erm)

This study

 pGE-KOamyE

pHY-ngCas9 derivative, 20 bp gRNA targeting amyE, 1000 bp donor DNA(deletion amyE)

This study

 pGE-PMspo0A

pHY-ngCas9 derivative, 20 bp gRNA targeting spo0A, 1000 bp donor DNA(point mutantion spo0A)

This study

 pGE-Iegfp

pHY-ngCas9 derivative, 20 bp gRNA targeting amyE, 1500 bp donor DNA(insetion P₄₃-egfp-T1 terminator)

This study

 pGE-KObpr

pHY-ngCas9 derivative, 20 bp gRNA targeting bpr, 1000 bp donor DNA(deletion bpr)

This study

 pGE-KOepr

pHY-ngCas9 derivative, 20 bp gRNA targeting epr, 1000 bp donor DNA(deletion epr)

This study

 pHY-SDFE27

pHY300PLK derivative, P_spovg-DFE27-T1 terminator

This study