CRISPR,CRISPR variant,Editing/Disruption Efficiency,Mechanism,Application,B. subtilis considerations,Year,Reference
Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system,CRISPR/Cas9,33% to 53% ,Assemble a complete CRISPR/Cas9 system in a single knockout plasmid which was build to each target. ,"Build a system to disrupt five genes (srfC, spoIIAC, nprE, aprE and amyE) in the B. subtilis ATCC 6051a genome. This application aims to control undomesticated properties that hamper the extracellular production of recombinant proteins.","B. subtilis ATCC 6051a has an endogenous plasmid pBS32 that encodes a single-pass transmembrane protein ComI, which hampers the transformation.",2016,https://www.nature.com/articles/srep27943
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Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis",CRISPRi  & CRISPR-Cas9,"~100% (single mutation)
~85% (double mutations)",Chromosonal expression of Cas9 and chromosomal transcription of sgRNAs using a separate gRNA transcription cassette with counterselectable sgRNA delivery vectors,A CRISPR/Cas9 toolkit to knockout or knockdown in single- or double-mutation/interference. This design obviates the need for multicopy plasmids and uses chromosomal integration for sgRNA transcripts and Cas protein. ,-,2016,https://pubmed.ncbi.nlm.nih.gov/27260361/
Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis,CRISPR-Cas9n ,"~80% (1-8kb gene deletions)
~100% (site-directed mutagenesis)
~23.6% (large fragment deletions)
~65% (three simultaneous point mutations)","Two-plasmid system which is composed of a vector enconding the CRISPR components  and another vector carrying the donor DNA. In addition, the ligD, a multifunctional non-homologous end joining DNA repair, was overexpressed. ",Develop an efficient and convenient method for modulating the B. subtilis multiplex (multiple genes) genome editing,The multiplex gene-editing system improved the editing efficiency of CRISPR/Cas9n mediated multiplexing by inhibiting nicks re-ligation. The gene ligD was targeted to regulate the nick repair mechanism.,2019,https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-019-1537-1
Design and Construction of Portable CRISPR-Cpf1-Mediated Genome Editing in Bacillus subtilis 168 Oriented Toward Multiple Utilities,CRISPR-Cpf1 ,"~100% (single mutation)
~59% (double mutations)","Cpf1 is able to process pre-crRNA to mature crRNA without tracrRNA. For multiplex genome editing, they employed the Cpf1 cassette integration in the genome and a plasmid with multi-crRNAs expression cassette. ","Build a CRISPR/Cpf1 platform for gene manipulation in B. subtilis, explore flexible deletion of a single gene and multiple genes or a gene cluster, and gene knock-in. In addition, Cpf1 showed decrease toxicity and off-targets.","The control of crRNA expression can improve cleavage efficiency. Pveg demonstrated crRNA high-level expression, which makes it difficult for bacteria to repair in time.",2020,https://www.frontiersin.org/articles/10.3389/fbioe.2020.524676/full
CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis,CRISPR/CAS9,editing efficiency of 76%,"Co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%","This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.",-,2019,https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210121
CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis.,CRISPRi ,-,"Several sgRNA expression cassettes (Pveg constitutive promoter) Genome integrate
 promoter) Genome integrate",Increase N-acetylglucosamine titer,-,2018,https://pubmed.ncbi.nlm.nih.gov/30176395/
CAMERS-B: CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis,CRISPR-Cpf1 ,"
~100% (single mutation)
~Proportion not shown (two and six mutations)","Two-plasmid system to express CRISPR/Cpf1 and the multi-crRNAs expression cassette. Otherwise, a Cpf1 modified was implemented to regulate gene repression. ","Build a CRISPR/Cpf1 platform for gene manipulation in B. subtilis, explore flexible deletion of a single gene and multiple genes or a gene cluster, and gene knock-in. In addition, dCpf1 assisted multiple genes regulation. ",-,2020,https://onlinelibrary.wiley.com/doi/full/10.1002/bit.27322?casa_token=heXpRkzd5mAAAAAA%3AckquQbAL6ffVkxbLiiFJtkJFbsr3Br_AAfDAywuFZKEkKXJdHfJpr-kO9cwVFqhZP8M3JYjV-Er2tjjKvQ
TA Systems,Toxin-Antitoxin,Type,Mechanism,,,,Reference
Toxin–Antitoxin Systems in Bacillus subtilis,Several,Type I & II,Endogenous Toxin-Antitoxin systems found in B. subtilis.,-,-,-,https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562991/
Sporulation,Spores Control,Type,Mechanism,Application,,,Reference
Bacterial Programmed Cell Death and Multicellular Behavior in Bacteria,skf-sdp,TA System type II,The proteins play a critical role in delaying sporulation by neighbours cell death.,-,-,,https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.0020135
Genetically Intact Bioengineered Spores of Bacillus subtilis,CRISPR/Cas9 digestion,-,Sporulation-inducible plasmid digestion,Genetically Intact Bioengineered Spores of Bacillus subtilis,-,,https://pubs.acs.org/doi/10.1021/acssynbio.0c00578
iGEM Applications,Kill Switch system,Type,Mechanism,Application,Test,Parts Registry,Reference
Marburg 2014,T4-Holin/ T4-antiholin + yvyD,TA system type II + ribosome inactivation ,"The system of three modules contains a T4-Antiholin/T4-Holin switch and yvyD toxin protein both regulated by the presence of IPTG. Without IPTG, the system triggers yvyD production to lysis the cell. In addition, a self-destruct module with Antiholin/holin enables lysis of the cells if a mutation occurs in the yvyD activation module. ",The system composed out of three interconnected modules which enable the survival of cells under laboratory conditions but no in the environment.,Math Modeling,-,http://2014.igem.org/Team:Marburg:Safety
Toulouse 2016,MazF/MazE + Zeta/Epsilon,TA system type II,"A designed system having two plasmids that must remain side-by-side in the cell or cause lysis if one of them gets out, both plasmids contains two differents Toxin-antitoxin device which is MazEF and Zeta/Epsilon.",Avoids horizontal gene transfer and spread the bacteria in the environment.  ,No,http://parts.igem.org/Part:BBa_K1937010,http://2016.igem.org/Team:Toulouse_France/Design#Confinement
Groningen 2016,CRISPR-Cas9,CRISPR,"A plasmid containing CRISPR-Cas9 device that would get activate to cleavage the circuit if no special treatment is applied. In this case, the deletion would be stopped with atc presence. ",This system was designed to prevent information leakage as well as address the issue of GMOs spreading ,No,-,http://2016.igem.org/Team:Groningen/Design
Freiburg 2016,MazF/MazE,TA system type II,"A late stage transcription factor regulator activates the MazF production. However, the team had some drawbacks in cloning this system, which was solved with a regulation of MazE production. The team modeled the killswitch design.  ",Prevents the spores form re-entering in the vegetative circuit. ,Math Modeling,-,http://2016.igem.org/Team:Freiburg/Killswitch
UNESP 2017,CRISPR/Cas9,Light-inducible CRISPR/Cas9 ,"The team combined a light-induced heterodimerzating proteins (CRY2 and CIB1) with split Cas9, which is activated by blue light and capable to cleavage different essential genes sites, as dnaN, rpoC and DNA Ligase genes. ",Release control of the organism in the environment.,No,-,http://2017.igem.org/Team:AQA_Unesp/Applied_Design
UNESP 2018,CRISPR/Cas9,Light-inducible CRISPR/Cas9 ,"The team combined a light-induced heterodimerzating proteins (nMag and pMag) with split Cas9, which is activated by blue light and capable to cleavage different essential genes sites, as dnaN, rpoC and DNA Ligase genes.",Release control of the organism in the environment.,No,"http://parts.igem.org/Part:BBa_K2660006
http://parts.igem.org/Part:BBa_K2660007
http://parts.igem.org/Part:BBa_K2660008
http://parts.igem.org/Part:BBa_K2660009
",http://2018.igem.org/Team:Unesp_Brazil/Design
UANL 2019,NucA/NucD/NucE,Light- or Arabinose-inducible nucleases expression,"The team designed two modules with an AND gate for NucE/NucA/NucD expression control by TetR. The first input is the pcyA/CcS/CcaR protein upstream to its inducible promoter, a green light-activated protein that regulates expression as a transcriptional factor, and the promoter pBAD, an arabinose-inducible system to trigger nucleases production when getting out of the presence of light and arabinose. ",Release control of the organism in the environment.,No,"http://parts.igem.org/wiki/index.php/Part:BBa_K3317063
http://parts.igem.org/wiki/index.php/Part:BBa_K3317062
http://parts.igem.org/wiki/index.php/Part:BBa_K3317066
http://parts.igem.org/wiki/index.php/Part:BBa_K3317053
http://parts.igem.org/wiki/index.php/Part:BBa_K3317052
http://parts.igem.org/wiki/index.php/Part:BBa_K3317069
",https://2019.igem.org/Team:UANL/Safeswitch
UANL 2020,-,-,"The team aimed to avoid sporulation process with spo0E expression to dephosphorylates spo0A~P, master regulator for entry into sporulation. ",Spores release control in the environment.,No,-,https://2020.igem.org/Team:FCB-UANL/Description