KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
Lysogeny broth (LB), Lennox	Fisher scientific	Cat# BP1427-2
Bacillus subtilis MC medium	Koo et al., 2017	N/A
Bacillus subtilis competence medium	Koo et al., 2017	N/A
IPTG	Denville scientific	Cat# C18280-13
Xylose
Ampicillin sodium salt	Sigma-Aldrich	Cat# A9518
Kanamycin sulfate	Sigma-Aldrich	Cat# K1377
Erythromycin	Sigma-Aldrich	Cat# E5389
Spectinomycin dihydrochloride pentahydrate	Sigma-Aldrich	Cat# S9007
Chloramphenicol	Sigma-Aldrich	Cat# C0378
Carbenicillin	Millipore-Sigma	Cat# 205805
Gentamicin sodium salt	Fisher Scientific	Cat# AAJ1605103
Trimethoprim	Sigma-Aldrich	Cat# T7883-5G
Q5 High-Fidelity DNA polymerase	New England Biolabs	Cat# M0493S
HiFi Assembly	New England Biolabs	Cat# E2621L
BsaI-HFv2	New England Biolabs	Cat# R3733
T4 DNA Ligase	New England Biolabs	Cat# M0202L
Critical Commercial Assays
DNeasy Blood & Tissue Kit	Qiagen	Cat# 69506
Midiprep Kit	Qiagen	Cat# 12143
QIAprep Spin miniprep kit	Qiagen	Cat# 27106
Deposited Data
Raw sequencing data (FASTQs) for relative fitness experiments and FACS-seq experiments	This study	SRA: PRJNA574461
Experimental Models: Organisms/Strains
Bacillus subtilis 168	BGSC	1A1
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm)	Peters et al., 2016	CAG74209
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), amyE∷Pveg-sgRNA(cat) (CRISPRi libraries: sgRNA spacers listed in Table S3)	This study	N/A
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), thrC∷Pveg-gfp(Spc)	This study	CAG78920
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), thrC∷Pveg-gfp(Spc), pJSHA77 (CRISPRi libraries: sgRNA spacers listed in Table S1)	This study	N/A
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), thrC∷Pveg-rfp(Spc)	This study	CAG78921
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), thrC∷Pveg-rfp(Spc), pJSHA77 (CRISPRi libraries: sgRNA spacers listed in Table S1)	This study	N/A
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), sacA∷Pveg-rfp	This study	CAG78922
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), sacA∷Pveg-rfp, murAA-gfp(Kan)	This study	CAG78923
Bacillus subtilis 168 lacA∷Pxyl-dcas9(Erm), sacA∷Pveg-rfp, murAA-gfp(Kan), thrC∷Pveg-murAA*(Spc)	This study	CAG78924
Escherichia coli BW25113	Baba et al., 2006	N/A
Escherichia coli BW25113 Tn7att∷PlLac-O1-dcas9(Gent)	This study	CAG78830
Escherichia coli BW25113 Tn7att∷PlLac-O1-dcas9(Gent), pJSHA77 (CRISPRi libraries: sgRNA spacers listed in Table S3)	This study	N/A
Escherichia coli BW25113 Tn7att∷PBBa_J23105-dcas9(Gent), yjaA:Pveg-gfp(Cat):yjaB	This study	CAG78108
Escherichia coli BW25113 Tn7att∷PBBa_J23105-dcas9(Gent), yjaA:Pveg-gfp(Cat):yjaB, pJSHA77 (CRISPRi libraries: sgRNA spacers listed in Table S1)	This study	N/A
Escherichia coli BW25113 Tn7att∷PBBa_J23105-dcas9(Gent), yjaA:Pveg-rfp(Cat):yjaB	This study	CAG78107
Escherichia coli BW25113 Tn7att∷PBBa_J23105-dcas9(Gent), yjaA:Pveg-rfp(Cat):yjaB, pJSHA77 (CRISPRi libraries: sgRNA spacers listed in Table S1)	This study	N/A
10-beta Electrocompetent Escherichia coli	New England Biolabs	Cat# C3020K
Oligonucleotides
Primers used in this study are listed in Table S9	This study	N/A
Recombinant DNA
pDG1731	Radeck et al., 2013	pBS4S (Addgene# 55170)
pDG1731-gfp	This study	N/A
pDG1731-rfp	This study	N/A
pDG1622	BGSC	ECE119
pJSHA77	This study	N/A
pJSHA77-rfp	This study	N/A
pJSHA77-gfp	This study	N/A
Software and Algorithms
Bowtie2	Langmead and Salzberg, 2012	http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
sgRNA design (fully matched sgRNAs)	This study	https://github.com/traeki/sgrna_design
Linear model training (train_linear_model.py)	This study	https://github.com/traeki/mismatch_crispri
Design a subset of mismatch sgRNA (choose_guides.py)	This study	https://github.com/traeki/mismatch_crispri
FASTQ analysis to calculate sgRNA abundance and relative fitness (count_guides.py, compute_gammas.py, gamma_to_relfit.py)	This study	https://github.com/traeki/mismatch_crispri
FlowJo v10	FlowJo, LLC
Other