Bacterial Genome Editing with CRISPR-Cas9 - ACS Publications

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Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable “Clean” Mutant Selection in Clostridium beijerinckii as an Example Yi Wang,†,‡,∇ Zhong-Tian Zhang,§ Seung-Oh Seo,†,‡ Patrick Lynn,∥ Ting Lu,‡,⊥ Yong-Su Jin,†,‡ and Hans P. Blaschek*,†,‡,# †

Department of Food Science and Human Nutrition, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ‡ Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States § Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ∥ Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ⊥ Department of Bioengineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States # The Integrated Bioprocessing Research Laboratory (IBRL), University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States S Supporting Information *

ABSTRACT: CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPRCas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable “clean” mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms. KEYWORDS: CRISPR-Cas9, genome engineering, synthetic biology, homologous recombination, single nucleotide modification (SNM), vector integration event (VIE)

Received: February 13, 2016

© XXXX American Chemical Society

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DOI: 10.1021/acssynbio.6b00060 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology

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vector integration event (VIE), which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on HR with plasmid-borne editing templates. We successfully employed CRISPR-Cas9 to select desirable “clean” mutants (containing desirable mutation without VIE or antibiotic marker). Besides, in this work, we also presented detailed development procedures for our customized CRISPRCas9 system, from screening proper promoters for Cas9/gRNA expression to constructing vectors by taking advantage of Cas9 in vitro digestion. Notably, the protocols and principles we developed in this study provide essential references for facilitating versatile genome editing in diverse microorganisms and general synthetic biology endeavors.

he capability of modifying microbial genome and thus conferring desirable phenotypes represents a great advancement in biotechnology. Metabolic engineering and synthetic biology methods keep improving and various genetic engineering tools have been developed over past decades. However, inevitable limitations exist with respect to these traditional approaches. For example, DNA addition based on nonreplicative integrational plasmids has generally low efficiency and meanwhile relies on integration of antibiotic markers for selection.1 DNA integration based on heterologous counter-selection markers or recombinase is usually time-consuming and laborious, and is also limited by the available selection markers and the expression of the recombinase in the host.2,3 Insertion-based mutagenesis (for example, with group II intron) can lead to polar effects and sometimes the mutation can reverse back due to the instability of the insertion.4,5 Furthermore, it is generally unfeasible to achieve high resolution “genome editing” using any of these traditional approaches. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system is an immune system in bacteria and archaea that can efficiently cleave foreign DNA entering the cells.6 Short DNA sequences located between CRISPR repeats make up a CRISPR array of targets. In the type II CRISPR-Cas system from Streptococcus pyogenes, the CRISPR array is transcribed to generate precursor CRISPR RNA (precursor crRNA), which is then matured by trans-activating small RNA, tracrRNA. The tracrRNA and pre-crRNA are afterward coprocessed by RNase III and make a dual-tracrRNA:crRNA, to guide Cas9 to cleave the target DNA.7 Recently, CRISPR-Cas9 has been explored as a leading-edge tool for genome engineering.8−10 Besides the dual-RNA configuration, a single chimeric guide RNA (gRNA, with features of both crRNA and tracrRNA) has been created and employed extensively to complex with Cas9 for genome engineering especially in eukaryotic hosts.8−11 It is interesting, however, that the application of CRISPR-Cas9, which is a tool derived from bacteria (or archaea), for genome engineering in bacteria rather dawdles. Initially, the natural CRISPR-Cas9 from S. pyogenes containing the dual-RNA and Cas9 was applied for genome engineering in Escherichia coli,12 S. pneumonia,12 and Lactobacillus reuteri.13 With the introduction of single or double strand linear DNA editing templates for homologous recombination (HR), all these cases relied on an established functional recombineering system or high recombinogenic capability in the host strains. Only until very recently, successes of genome engineering using CRISPR-Cas9 have been reported in Streptomyces species,14−16 Tatumella citrea,17 and Clostridium cellulolyticum18 (using Cas9 nickase, a Cas9 mutant which leads to breakage on only one strand of the chromosome11). In this study, we took C. beijerinckii as a representative to explore CRISPR-Cas9 for genome engineering in microorganisms that lack developed genome engineering tools. C. beijerinckii has great industrial significance for producing valuable biofuel and biochemicals from various carbon sources.19−21 As an anaerobic spore-forming Gram-positive bacterium, it is known for the difficulty to be metabolically engineered and the complexity of its physiology.22 Recently, we reported our preliminary results in markerless chromosomal gene deletion in this microorganism using S. pyogenes CRISPR-Cas9 system.23 Here, taking a further step, by combining inducible expression of Cas9 and plasmidborne editing templates, we successfully achieved multiplex genome editing purposes, including gene deletion, DNA integration and single nucleotide modification (SNM) with very high efficiency. During our experiments, we observed severe

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RESULTS AND DISCUSSION Establish gRNA Expression in C. beijerinckii. Since the expression and functionality of the natural dual-RNA guiding Cas9 system from S. pyogenes was hard to predict in C. beijerinckii, we decided to customize the chimeric single gRNA guiding Cas9 system for proper expression using native promoters from C. beijerinckii. Supposing that the expression of Cas9 ORF (which is from a Gram-positive bacterium and has a low GC content (35% GC); GC content in C. beijerinckii is about 30%) is expected to be straightforward, we first focused on establishing efficient expression of the chimeric gRNA in C. beijerinckii. On the basis of our RNA-Seq data,24,25 ORF promoters with various strengths were selected for screening to drive gRNA expression. Since the gRNA is a noncoding small RNA (sRNA), promoters driving sRNA expression in C. beijerinckii with high strengths were included in this screening. It has been reported that the mammalian RNA polymerase III U6 promoter can efficiently drive the mammalian expression of tracrRNA;9,26 thus the RNA polymerase (Cbei_0144) promoter from C. beijerinckii has also been included. The sqRT-PCR was conducted and a band of 76 bp would be expected if there was effective gRNA expression. As illustrated in Figure 1b, out of the eight promoters investigated, visible PCR bands were observed for six of them, with the construct containing sRNA sCbei_5830 promoter generating the brightest (Lane #11 in Figure 1b). This was surprising but not unreasonable since the chimeric gRNA itself is a noncoding sRNA. Therefore, this promoter was selected for gRNA expression to construct the functional CRISPR-Cas9 system. Lethality of Double-Strand Breakages (DSBs) Induced by Active Cas9. It has been reported that CRISPR-Cas9 mediated cleavage can induce recombination in cells and introduction of editing template DNA fragment carrying designed mutations can lead to the desirable mutations.10,12 Attempts were first carried out in this study to obtain desirable mutations through cotransformation of linear DNA editing template along with the CRISPR-Cas9 vector. A series of editing templates were designed such that a 50 bp including the 20-nt protospacer targeting sequence (contained in pYW19-pta) was deleted and various lengths of arms from 0.1 kb to 2 kb flanked at both sides of the deletion region (ptaE1-ptaE6, Table S2). Co-transformation of pYW19-pta along with one specific editing template was initially conducted (1 μg of vector along with 1 μg of editing template), and no colonies were obtained. While colonies could be consistently obtained from the control transformation employing pYW19-BseRI (along with the same editing template used in transformation with pYW19-pta) with a transformation efficiency of around 4.17 × 102 cfu/μg-DNA. Further, higher concentrations of editing templates and vector were employed (up to 10 μg along with up to 2.5 μg of pYW19-pta) to allow for a better HR B


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Figure 1. Screening of promoters for gRNA expression. (a) The scheme of pYW19gRNA-BseRI, a general vector used to screen various promoters for gRNA expression. Two BseRI sites have been included upstream of the gRNA sequence. The designed promoter sequence can be amplified using PCR and inserted upstream of gRNA through Gibson Assembly after the vector was digested with BseRI. (b) The expression of gRNA with various promoters has been tested with semiquantitative reverse transcription PCR (sqRT-PCR). Promoter tested for the gRNA expression: Lane #1: Cbei_0144 (RNA polymerase promoter; 284 bp); Lane #3: sRNA sCbei_2478; Lane #5: Cbei_0075; Lane #7: Cbei_1823; Lane #9: Cbei_2561; Lane #11: sRNA sCbei_5830; Lane #13: sRNA sCbei_0761; Lane #15: Cbei_0144 (RNA polymerase promoter; used all the 330 bp of the intergenic region between Cbei_0143 and Cbei_0144). The even number lane is the corresponding negative control (PCR with corresponding RNA as template) to the lane on its left (for example, Lane #2 is the negative control for Lane #1, Lane #4 is the negative control for Lane #3, and so on). Lane P: Positive control (PCR with plasmid DNA as template; the gRNA expression plasmid with Cbei_0075 promoter has been used here). Lanes MS: 100 bp DNA marker, with numbers on the right corresponding to the marker length in bp (NEB).

Actually, the lethality of CRISPR-Cas9 system to bacterial strains has been repeatedly reported by other researchers.14−16,18 In eukaryotic systems, highly efficient endogenous NHEJ can achieve the repairing of DSB and sometimes lead to mutations. In prokaryotes, many microorganisms lack the NHEJ mechanism, or the NHEJ is not efficient enough to achieve the repairing of DSB induced by Cas9, and thus normally DSB occurring on the single copy of chromosome leads to cell death. Furthermore, the linear DNA fragment that was cotransformed along with CRISPR-Cas9 vector was not able to repair DSB either.

opportunity. However, no colony was obtained in any of these attempts either. Therefore, we tentatively concluded that the DSB induced by Cas9 is lethal to the cells and cannot induce effective recombination in C. beijerinckii to achieve the DSB reparation; meanwhile, the endogenous nonhomologous endjoining (NHEJ) in C. beijerinckii is not efficient either to achieve repair of the DSB. To further confirm our hypothesis, we targeted on another site (5′-GCAGAAAAAATACAAAAATT-3′) with the transformation of pYW19-pta2. Similarly, no colony was obtained (Figure S1). C


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mutant cells against the nonedited background cells. A mutation rate (number of mutants out of total transformants) of 100% was achieved; however, the transformation efficiency (cfu/μg-DNA) was extremely low due to the low HR efficacy in C. beijerinckii and strong selection power by Cas9. Therefore, this approach was still unsatisfying for reliable genome editing purposes (in many cases, we hardly got colonies out of the transformation). With respect to the CRISPR-Cas9 construct, we creatively applied a sporulation gene promoter to drive Cas9 expression and thus gave time for HR to occur before DSB.23 We further hypothesized that, if we would apply an inducible promoter for Cas9 expression and allow enough time for HR to occur before inducing Cas9 expression, we would have an even greater chance to obtain the desirable mutants using CRISPR-Cas9 selection. A lactose inducible promoter originally developed for C. perf ringens27 had been reported to be functional in C. acetobutylicum when it was used to develop an inducible counter-selection marker for genetic engineering purposes.2 Here, we employed the lactose inducible promoter to drive the Cas9 expression in C. beijerinckii, and a similar subculturing procedure as reported by Al-Hinai et al.2 was employed to enforce HR. After transformation with pYW34-ptaE3, no colonies were obtained from TGYLE25, which was not surprising due to the powerful selection pressure of Cas9. Colonies were obtained from TGYE25 with a transformation efficiency of 1.05 × 102 cfu/μg-DNA. After serial subculturing and replating onto TGYLE25, positive mutants were identified using cPCR as we did for the transformation with pYW27-ptaE3. Eight colonies were tested and all of them were confirmed as positive mutants (data not shown). Large Gene Fragment Deletion. Next, we attempted to achieve larger fragment deletion using the inducible CRISPR-Cas9 system. Vector pYW34-ptaE7 was designed to delete 1.5 kb (936 bp of pta ORF, 96 bp intergenic region and 468 bp of acetate kinase (ack) ORF) from the chromosomal of C. beijerinckii (Figure 2c). After transformation, one colony was obtained from TGYLE25 while a transformation efficiency of 3.94 × 102 cfu/μg-DNA was achieved with TGYE25. The colony from TGYLE25 was tested with cPCR using primers P50 and P51 and confirmed as a positive mutant (Figure 2d). Study is underway in our lab to test for even larger gene fragment deletion in C. beijerinckii using our customized CRISPR-Cas9 system. Recently, large gene fragments of up to 30 kb in Streptomyces strains14 and 133 kb in E. coli28 have been successfully deleted using the CRISPR-Cas9 based systems with various strategies. Markerless Chromosomal Gene Integration. Using the CRISPR-Cas9 system, we can also obtain markerless gene fragment integration (allelic exchange). The vector pYW34-ptaE8 was designed to insert C. beijerinckii adhE (along with native promoter and terminator, a total of 1614 bp) into the pta locus to replace part of pta ORF (849 bp) (Figure 3a). After transformation, no colony was obtained from TGYLE25 while colonies were obtained from TGYE25 (with a transformation efficiency of 2.92 × 102 cfu/μg-DNA). After a series of subculturing and replating on TGYLE25, cPCR results with primers P26 and P57 confirmed that 15 out 16 picked colonies demonstrated positive mutant bands (Figure 3b). Single Nucleotide Modification (SNM). We next attempted to achieve even finer genome editing using our CRISPR-Cas9 system, that is SNM. Technically, SNM can be achieved with CRISPR-Cas9 if the locus to be edited is within the “GG” loci of a Protospacer Adjacent Motif (PAM) or the 20-nt protospacer whenever the SNM can efficiently disrupt the

This demonstrated that the recombination efficiency in C. beijerinckii is not high enough to achieve the reparation of DSB as is the case in various eukaryotic systems. In addition, the transformed linear DNA was not replicable, and the host exonuclease system in C. beijerinckii might have accelerated their rapid degradation. On the other hand, it has been reported that if a string of six or more Ts (or As) is contained in the 20-nt gRNA sequence, the targeting of chromosome by gRNA may not be efficient.10 Interestingly, in pYW19-pta2, even though there were two strings of >6 As/Ts in the guiding sequence region, the transformation of this vector still led to severe cell death. Achieve Genome Editing Using CRISPR-Cas9 and Plasmid-Borne Editing Template. Since cotransformation of CRISPR-Cas9 along with linear DNA editing templates simply led to cell death, no successful genome editing could likely be achieved with this approach. In the further attempt, rather than using Cas9 as a mean to induce recombination as demonstrated in many eukaryotic cells, we attempted to use Cas9 as a selection tool. We put the DNA editing template onto the same vector as Cas9 and gRNA sequences with the expectation that a HR event would occur through a double-crossover event.2 Therefore, by functioning as a selection tool against nonedited cells, the CRISPR-Cas9 system was expected to confer a high efficiency for genome engineering in favor of the positive mutants induced by HR.23 To test this hypothesis, a 2 kb fragment ptaE3 (50 bp including the 20-nt protospacer from pta ORF was deleted with 1 kb homologies flanking at both sides; Table S2) was integrated into the CRISPR-Cas9 vector. Meanwhile, in order to allow time for cells to experience HR before the Cas9 selection (brought about by DSB on the target site), a sporulation gene spoIIE (Cbei_0097) promoter was employed based on our RNA-Seq data (Figure S2) to drive the Cas9 expression such that upregulated Cas9 expression was only induced at a late stage of cell growth.23 Vector pYW27-ptaE3 or the control vector pYW27-BseRI-ptaE3 was transformed into C. beijerinckii through electroporation. After ∼48 h of growth on TGYE25, five colonies were observed from the transformation with pYW27-ptaE3 while a transformation efficiency of 4.49 × 102 cfu/μg-DNA was obtained for the control vector. Colony PCR (cPCR) was conducted to confirm the mutation in the transformants. A pair of primers P26 and P27 (Table S2) flanking the upstream and downstream, respectively, of the HR targeting region (and thus the primers can only anneal to the chromosome but not the vector) were used for the test. While it was difficult to tell the size difference between PCR bands from positive transformants (expected as 3000 bp) and those from the wild type (3050 bp), the generated PCR product from cPCR was gel-extracted, and an additional PCR was carried out using the purified product as template with primers (P41 and P42) targeting on a shorter sequence that directly flanked the 50 bp deletion region. Results confirmed the desirable deletion for all five transformants from pYW27-ptaE3 (Figure 2b). The mutation was further verified by Sanger sequencing. Additional cPCR with the same procedure was conducted for the transformants with the control vector, and results demonstrated that all of them generated the same band size as the control using wild type gDNA as template (data not shown). Similar strategy for the deletion of a fragment in spo0A gene was reported in our previous study;23 here this strategy was further validated by deleting another gene fragment with a similar efficiency. Achieve Reliable Genome Editing by Expressing Cas9 with an Inducible Promoter. By this stage, successful genome editing was achieved through Cas9 selection in favor of the D


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Figure 2. Gene deletion using the customized CRISPR-Cas9 system. (a) pYW27-ptaE3 was designed to delete 50 bp of pta ORF. Bars in green represent the homologous recombination (HR) arms either on the chromosome or on the plasmid; the short bar in red represents the 50 bp to-be-deleted DNA fragment. (b) Colony PCR (cPCR) results demonstrated the desirable deletion. Two rounds of PCR have been conducted, and the results from the second round were shown here. First, primers P26 and P27 flanking the upstream and downstream respectively of the HR targeting region (thus only annealing to the chromosome but not the vector) were used to screen the positive mutants (wild type genomic DNA (gDNA) as control). Then the generated PCR product from cPCR was gel-purified, and second round of PCR was carried out using the purified product as template with primers (P41 and P42) that directly flanked the 50 bp deletion region. Lanes MS: 100 bp DNA marker, with numbers on the left corresponding to the marker length in bp (NEB); Lane 1: using the gel-purified cPCR product as template (size: 242 bp); Lane 2: using the C. beijerinckii 8052 gDNA as template (size: 292 bp). (c) pYW34-ptaE7 was designed to delete 1.5 kb of chromosome. Bars in green represent the HR arms either on the chromosome or on the plasmid; the bar in red represents the 1.5 kb to-be-deleted DNA fragment. (d) cPCR with primers P50 and P51 flanking the upstream and downstream respectively of the HR targeting region (thus only annealing to the chromosome but not the vector) confirmed the expected 1.5 kb deletion. Lanes ML: 1 kb DNA marker, with numbers on the left corresponding to the marker length in kb (NEB); Lane 3: directly picked mutant colony as template (size: 3381 bp); Lane 4: using the C. beijerinckii 8052 wild type genomic DNA as template (size: 4881 bp). spoIIEP: the promoter of spoIIE gene (Cbei_0097); Cas9: Streptococcus pyogenes Cas9 ORF amplified from the plasmid pMJ806 (purchased from Addgene);11 thlT: terminator of thiolase gene (Cbei_0411); sRNA-P: promoter of the small RNA gene (sCbei_5830);37 gRNA: the chimeric single guide RNA,8 with a 20-nt guiding sequence (5′-GATGCAGATGGAATGGTATC-3′) targeting on the pta ORF; tracrRNA-T: transcription terminator derived from S. pyogenes;36 CAK1: C. beijerinckii Gram-positive replicon; Ermr: the erythromycin resistant marker; lacP: the lactose inducible promoter.2

recognition by Cas9 on the original target site.12 If the CRISPRCas9 system has high enough specificity, when a SNM is made either in the PAM or the 20-nt protospacer (through HR), the mutant can be selected based on the same mechanism as we described above for gene deletion or integration. However, if the desirable SNM is not in a PAM or the protospacer sequence upstream of a PAM, this strategy cannot be applied. Here, we employed a two-step strategy to achieve SNM to overcome the restriction by PAM availability. As shown in Figure 4a, we attempted to change “GAA” to “GAT” (E to D) within pta ORF where there was no PAM in range that we could take advantage of in order to use the above-described strategy. Through a first step, with the transformation of pYW34-ptaE9, we created an artificial PAM (aPAM) at the targeted locus by changing “GAA” to “GGG” and meanwhile changed the PAM (PAM1) we used in this transformation from “AGG” to “ATT” (named as modified PAM1, or mPAM1) for Cas9-assisted selection purpose. Then in a second step, through the transformation with pYW35-ptaE10 using aPAM (“GGG”) for the targeting purpose, we changed the mPAM1 back into the original “AGG” (PAM1) and meanwhile changed the aPAM to the desirable “GAT”. Through such a twostep strategy (Strategy I), we successfully achieved the desirable SNM, which has been confirmed with both SURVEYOR

Mutation Detection assay (IDT) (Figure 4d) and Sanger sequencing (Figure 4e). For the transformation with pYW35ptaE10, a second antibiotic marker (Sp marker) was employed to avoid the laborious plasmid curing process for transformants obtained in the first step.29 SNM represents the finest genome editing endeavor. As demonstrated above, with our two-step strategy (Strategy I) during which an artificial PAM is created in the intermediate step, we can virtually achieve SNM at any desirable locus overcoming the limitation of PAM availability. Furthermore, an alternative two-step approach (Strategy II) for SNM has been developed during which a gene deletion is first obtained as an intermediate step, which is even more advantageous and preferable. Such a strategy allows easier detection of the mutation through cPCR and meanwhile likely alleviates VIE. In Strategy II, we however still need to create an artificial PAM (for the Cas9 targeting purpose in the second step) besides the gene fragment deletion. Actually, this could be avoided by employing the same PAM of “NGG” (but different 20-nt protospacer) for the deletion and integration steps to achieve desirable SNM as illustrated in Figure 4c. Because an appropriate PAM can almost always be found within a distance of less than couple of kilobases (the length that we tested in this study to effectively achieve E


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without VIE, we ran cPCR for >300 colonies for both mutants). Interestingly, none of the VIE was likely obtained through a simple “single crossover” event; rather, all the mutants containing VIE (or VIE mutants) contained two copies of the editing template between which the other elements from the original plasmid were integrated. No fragment containing the gRNA targeted region was detected. This further demonstrated the strong selection power of CRISPR-Cas9. As shown in Figure 5 (using gene deletion as an example), the genome engineering may have been achieved through a two-step process (double-crossover).30 In the first step, plasmid integration occurred via a single-crossover with either the up- or downstream homologous region on the chromosome, and could potentially lead to two architectures. In the second step, another recombination occurred and further led to two potential architectures, within which Type B was the final desirable mutant. However, the CRISPR-Cas9 vector was replicative and as long as copies of CRISPR-Cas9 vectors were still present in the cell, additional single-crossover could further occur and lead to integration of plasmid into the chromosome and generate an architecture of Type C. Therefore, finally, the “positive” mutant we obtained through screening with cPCR was actually a mixture, Type C cells containing VIE and Type B cells with clean mutation. The cPCR results only demonstrated the amplicons from the desirable mutants (Type B cells), because the ones from Type C cells would be longer than 15 kb and thus beyond the cPCR detection capability. Of course, in the scheme discussed above, Type A cells could also experience additional crossovers and generate more complicated architectures containing integrated plasmids. However, as long as the gRNA targeted region was included in Type A and its derivatives, these mutants will not be able to survive at the induction of Cas9. Therefore, this case (further recombination of Type A) will not be further discussed here. Alternative Approach for SNM. With the developed CRISPR-Cas9 system, we successfully achieved SNM. However, in the transformation with pYW34-ptaE9 and pYW35-ptaE10, severe VIE occurred which led to a laborious process in order to identify a desirable clean mutant. In addition, in both transformations, since only several nucleotides were changed, it was impossible to identify positive transformants solely based on the size differences of PCR bands. Therefore, we further developed an alternative two-step approach (Strategy II) for SNM (Figures 5B,F). In pYW34-ptaE11, the editing template was designed to delete 200 bp (including the 20-nt protospacer and PAM) from the chromosome and meanwhile an aPAM was created as was done in ptaE9 (by changing “GAA” to “GGG”). Then in a second step with transformation using pYW35-ptaE10, the aPAM (along with the upstream 20-nt) was used for the Cas9 targeting purpose and was altered into desirable “GAT”. Therefore, through such a strategy, a same SNM (“GAA” to “GAT”) was achieved as described above. In the first step for transformation with pYW34-ptaE11, following serial transfers and lactose-induction procedure, positive colonies were identified using cPCR. One positive colony was subjected to replating and detection for VIE. Nine out of 17 tested colonies were confirmed as clean mutants without VIE. One of the positive mutants was cultured and used for the transformation with pYW35-ptaE10. Finally, a positive mutant with SNM was identified and confirmed by Sanger sequencing. VIE were tested after replating; 7 out of 23 colonies that had been tested were clean mutants.

Figure 3. Chromosomal gene integration (allelic exchange) using the customized CRISPR-Cas9 system. (a) The vector pYW34-ptaE8 was designed to insert adhE (along with native promoter and terminator, totally 1614 bp) into the pta locus to replace part of the pta ORF (849 bp). Bars in green represent the homologous recombination (HR) arms either on the chromosome or on the plasmid; the bar in yellow on the plasmid represents the to-be-integrated adhE gene fragment; the bar in red on the chromosome represents the to-be-replaced gene fragment by adhE integration. lacP: the lactose inducible promoter;2 Cas9: the Streptococcus pyogenes Cas9 ORF amplified from the plasmid pMJ806 (purchased from Addgene);11 thlT: terminator of thiolase gene (Cbei_0411); sRNA-P: the promoter of the small RNA gene (sCbei_5830);37 gRNA: the chimeric single guide RNA,8 with a 20-nt guiding sequence (5′-GATGCAGATGGAATGGTATC-3′) targeting on the pta ORF; tracrRNA-T: the transcription terminator derived from S. pyogenes;36 CAK1: the C. beijerinckii Gram-positive replicon; Ermr: the erythromycin resistant marker. (b) Colony PCR (cPCR) with primers P26 and P57 (Table S2) flanking the upstream and downstream respectively of the homologous recombination targeting region (thus only annealing to the chromosome but not the vector) confirmed the expected gene integration. Lanes ML: 1 kb DNA marker, with numbers on the left corresponding to the marker length in kb (NEB); Lane 1: directly picked mutant colony as template (PCR amplicon size: 3879 bp); Lane 2: using the C. beijerinckii 8052 genomic DNA as template (PCR amplicon size: 3114 bp).

deletion or integration in a single step; however, longer deletion or integration may also work and warrants further investigation) from the desired locus for SNM, such a strategy (Strategy III) would make the SNM even easier achievable. Vector Integrating Problem. Since the Cas9-assisted genome engineering achieved here was essentially through HR, we attempted to test whether it was possible that the whole plasmid had been integrated into the chromosome through “single crossover”.23 Originally identified “positive” mutants were subjected to replating to fresh TGY plates. Single colonies were then picked and cPCR was carried out using a forward primer annealing to the upstream chromosome and a corresponding reverse primer annealing to the plasmid (for testing the upstream joint between the chromosome and plasmid) or using a forward primer annealing to the plasmid and a corresponding reverse primer annealing to the downstream chromosome (for testing the downstream joint between the plasmid and chromosome). Surprisingly, the results indicated that, more or less, VIE occurred in almost all the mutants that were previously identified as “positive” (Table S3). Specifically, for mutants from pYW27-ptaE3, 10 out of the 15 tested colonies had VIE. For mutants from pYW34-ptaE3, 12 out of the 15 tested colonies had VIE. For the mutant from pYW34-ptaE7, no VIE was detected from the replating (the positive mutant was obtained from the only colony that grew on TGYLE25 after transformation). For mutants from pYW34-ptaE8, 13 out of 15 tested colonies had VIE. While for mutants from pYW34-ptaE9 and pYW35-ptaE10, more than 99% of the tested colonies from the replating had VIE (in order to obtained the positive mutant F


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ACS Synthetic Biology Using CRISPR-Cas9 as a Tool for Positive Mutant Selection. As discussed above, during the genome engineering process using CRISPR-Cas9, more or less VIE occurred depending on different engineering purposes. The process for identifying clean mutants without VIE was time-consuming and arduous. We noticed the VIE problem in our previous report,23 but we were not able to study the mechanism in detail and did not find a way to address the problem at that time. Here, we further explored CRISPR-Cas9 as a tool for selecting positive clean

mutants by eliminating VIE mutants. Vector pYW35-Erm containing Sp as the selection marker was designed to have the Cas9 targeting on the Erm marker region. Therefore, the mutant with integrated vector containing the Erm marker would be removed from the mixture by the Cas9-induced DSB and the clean mutant could be selected. The mixed transformants (containing clean mutants and VIE mutants) obtained from transformation with pYW34-ptaE8 was cultured and selected with pYW35-Erm. The cPCR results confirmed that all tested

Figure 4. continued G


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Figure 4. Single Nucleotide Modification (SNM) using CRISPR-Cas9. (a) A two-step strategy (Strategy I) changing “GAA” to “GAT”. With pYW34ptaE9, an artificial PAM (aPAM) was created and the PAM (PAM1) used in this transformation was changed to “ATT” (mPAM1). With pYW35-ptaE10 using aPAM for targeting, mPAM1 was changed into the original “AGG” and aPAM was changed to “GAT”. (b) An alternative two-step strategy (Strategy II) changing “GAA” to “GAT”. With pYW34-ptaE11, an aPAM was created and 200 bp was deleted. With pYW35-ptaE10 using aPAM for targeting, the deleted 200 bp was integrated into the original locus and aPAM was changed to “GAT”. (c) Another proposed two-step strategy (Strategy III) for SNM. Suppose that “X” at random locus is desired to change to “Y”. In the first step, “20N” in pink with PAM which could be several bp to several kb from targeted locus is selected for the targeting purpose. After transformation, fragment including sequence from protospacer (full or partial, as long as Cas9 targeting can be disrupted) to targeted locus (or even farther) is deleted. In the second step, using “20N” in black with the same PAM for Cas9 targeting, the deleted sequence (“X” modified to “Y”) is integrated into the original locus. (d) SNM confirmation with SURVEYOR mutation detection assay. 1050 bp fragments were amplified from transformants of pYW35-ptaE10 (corresponding to Lanes 1−4) and pYW34-ptaE9 (corresponding to Lane 5); same PCR but with wild type (WT) gDNA as template generated Reference DNA. Lanes 1−4: SNM from Reference DNA, and thus three bands appeared: 305, 745, and 1050 bp (yellow triangles). Lane 5: Two mismatches from Reference DNA, and thus six bands appeared: 208, 305, 513, 537, 745, and 1050 bp (blue triangles; 513 and 537 bp could not be separated). Lane MS: 100 bp marker (NEB). (e) Sanger sequencing confirmation of SNM. (f) Electrophoresis confirmation of SNM through Strategy II. Lane 6: WT gDNA as template (2280 bp). Lane 7: pYW34-ptaE11 transformant gDNA as template (2080 bp). Lane 8: pYW35-ptaE10 transformant gDNA as template (2280 bp). All three PCRs were conducted with primers P86 and P85. Lanes ML: 1 kb marker (NEB).

(containing Sp marker whose Cas9 targeted on Erm marker of the integrated vector), the obtained Sp resistant transformants were replated onto TGYE25, and no colony was obtained. This indicated that the transformation of pYW35-Erm also led to curing of the original CRISPR-Cas9 vector pYW34-ptaE8 besides elimination of the integrated vector. However, on the other hand, since pYW35-Erm and pYW34-ptaE8 both contained the same origin of replication (they were both pTJ1 derivatives), the transformation of pYW34-Sp could also have simply led to the pop-out of pYW34-ptaE8.29 To remove the VIE mutants, the expression of Cas9 could be constitutive rather than inducible; therefore, a strong constitutive promoter can be employed for this purpose. During construction of our vectors, we successfully applied Cas9 for in vitro digestion (see Supporting Information), and therefore overcame the strict dependency on restriction enzyme sites as in the case for traditional DNA cloning. Along with the advancement of DNA de novo synthesis technology and the molecular cloning technology (such as Gibson Assembly,33 Gateway cloning,34 etc.), the in vitro application of Cas9 nuclease enables metabolic engineers and synthetic biologists as they wish to achieve sequence-independent, scarless and seamless cloning purposes. Conclusions. Here we reported highly efficient genome editing including gene deletion, gene integration in C. beijerinckii achieved in single steps by combining inducible expression of Cas9 and plasmid-borne editing templates. We also attained

colonies contained no integrated vector. Further cPCR verified that these were all positive mutants (with adhE gene integrated; data not shown). On the other hand, the vector pYW34-Sp containing Erm as the selection marker was designed to have the Cas9 targeting on the Sp marker region. Therefore, the mutant with integrated vector containing Sp marker would be removed from the mixture and the clean mutant could be selected. The mixed transformants (containing clean mutants and VIE mutants) obtained from transformation with pYW35-ptaE10 (with Strategy I) were transformed and selected with pYW34-Sp. The cPCR results confirmed that all tested colonies contained no integrated vector. Sanger sequencing verified that these were all positive mutants (with SNM; data not shown). We demonstrated that CRISPR-Cas9 can be employed as a tool to select the clean mutant out of the mixture by eliminating VIE mutants. This suggested that CRISPR-Cas9 can be used as an efficient tool for selectively removing undesirable microorganisms from a microbial community. Indeed, recently several research groups have reported their application of CRISPR-Cas9 system for programmable removal of bacterial strains.31,32 Meanwhile, the effort to eliminate VIE mutants using CRISPRCas9 by targeting on the integrated vector could also have removed the original transformed CRISPR-Cas9 vector that contained the same target site; therefore, further recombination of the vector and the edited region on the chromosome could be avoided. Actually, during our experiment for the isolation of “clean” SNM mutants with transformation of pYW35-Erm H


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Figure 5. Schematic presentation of possible mechanism for the vector integration event (VIE) during genome engineering with CRISPR-Cas9 (taking gene deletion as an example). The genome engineering might have been achieved through a two-step process (double-crossover). In Step 1, plasmid integration occurred via a single-crossover recombination with either the up- or downstream homologous region on the chromosome, and could potentially lead to two architectures. In Step 2, another recombination event occurred and further led to two potential architectures, within which Type B was the final desirable clean mutant. However, the CRISPR-Cas9 vector was replicative and as long as copies of the original CRISPR-Cas9 vectors were still present in the cell, additional single-crossover could further occur (Step 3; need to notice that either fragments (represented with green and cyan color respectively) in Type B can experience additional single-crossover with its corresponding homology in the CRISPR-Cas9 vector, but does not necessarily occur at the same time) and lead to the integration of plasmid into the chromosome and generate an architecture of Type C. Therefore, finally, the “positive” mutant we obtained through screening with cPCR was actually a mixture, including the Type C cells containing VIE and the Type B cells with clean desirable mutation.

transformation was conducted through electroporation as described previously,4 except that 15% glycerol has been used as the washing buffer in place of polyethylene glycol 8000 (PEG 8000; Sigma-Aldrich, St. Louis, MO). All the DNA oligonucleotide sequences used in this study are listed in Table S2 in the Supporting Information. The linear DNA fragments designed for DSB repairing were amplified through a combination of regular PCR and Overlap Extension (SOEing) PCR using appropriate primers as indicated in Table S2. Plasmid Construction. In order to screen for the proper promoter for the expression of chimeric gRNA in C. beijerinckii, a series of vectors were constructed containing respectively various promoters followed downstream by the chimeric gRNA sequence.8 To facilitate the construction of the series of vectors, a general vector was first constructed as follows. A 457 bp gBlock fragment (gBLK01, see Table S2) was synthesized by Integrated DNA Technologies (IDT), including a partial sequence of the thiolase gene (Cbei_0411) terminator (thlT), a 45 bp random sequence containing two BseRI sites fused with the gRNA sequence,8 and a transcription terminator derived from S. pyogenes.36 Using this gBlock as template, the inset fragment was amplified using primers P01 and P02 (Table S2). Then the amplified fragment was inserted into pTJ1 between the ApaI and NotI restriction enzyme sites through Gibson Assembly,33

SNM by applying innovative two-step approaches, which eliminated the strict reliance on the availability of PAM sequences. During our genome editing process, we observed severe VIE, which we think is difficult to avoid but has never been reported by other researchers for the similar studies. We further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable “clean” mutants by eliminating the VIE mutants. The principles and approaches we reported herein are broadly applicable and will provide essential references to other researchers for precise genome editing in diverse microorganisms.

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METHODS Strains, Culture Conditions, Plasmids, and Oligonucleotides. All bacterial cultures and plasmids used in this study are listed in Table S1 in the Supporting Information. E. coli transformants were grown aerobically at 37 °C in LB medium supplemented with 100 μg/mL of ampicillin or 50 μg/mL of kanamycin as appropriate. C. beijerinckii 8052 wild type strain and transformants were grown anaerobically at 35 °C in tryptoneglucose-yeast extract (TGY) medium containing 30 g/L of tryptone, 20 g/L of glucose, 10 g/L of yeast extract, and 1 g/L of L-cysteine, supplemented with 25 μg/mL of erythromycin (Erm) or 750 μg/mL of spectinomycin (Sp) as needed.4,35 Clostridial I


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the outgrowth in TGY medium, the cells were spun down and resuspended in 300 μL of fresh TGY. One hundred microliters of the cell suspension was spread onto a TGY plate (one plate) supplemented with 25 μg/mL of Erm (TGYE25) (or a TGY plate supplemented with 750 μg/mL Sp (TGYSp750) when Sp marker was used) and TGY plates (two plates) supplemented with 40 mM lactose and 25 μg/mL of Erm (TGYLE25) (or TGY plates (two plates) supplemented with 40 mM lactose and 750 μg/mL Sp (TGYLSp750) when Sp marker was used). The plates were incubated in an anaerobic chamber at 35 °C until colonies were observed (usually 24−48 h). Putative mutant colonies growing on TGYLE25 (or TGYLSp750) were isolated and screened by cPCR. If no colonies (in most cases) were observed on the lactose supplemented plates, colonies from TGYE25 (or TGYSp750) plates were picked and inoculated into liquid medium (that is, TGY containing appropriate antibiotics). A series of subculturing (with a 5% v/v inoculum) was carried out to enrich the desirable HR. After five rounds of transfers, a 5% v/v inoculum from the last culture was transferred to fresh TGY medium supplemented with 40 mM lactose and appropriate antibiotics and allowed to grow for additional 5−6 h. Serial dilutions of the culture were then plated onto TGYLE25 (or TGYLSp750), and the plates were incubated until colonies were observed. Putative mutant colonies were then screened with cPCR. Confirmation of SNM Using Mutation Detection Assay. Colonies from transformation with pYW35-ptaE10 and from transformation with pYW34-ptaE9 were subjected to cPCR with primers P26 and P27 (Table S2; with amplicon size of 3050 bp). Then the PCR products were gel-purified, and subjected to second round of PCR with primers P30 and P31 (Table S2; with amplicon size of 1050 bp); meanwhile, PCR using C. beijerinckii gDNA as template (with primers P30 and P31) was conducted to get the product as Reference DNA for the detection. The 1050 bp PCR products were cleaned up and used for the point mutation detection using SURVEYOR Mutation Detection kit (IDT) following the manufacturer’s protocol. Each PCR product from mutants was mixed respectively with the Reference DNA at equal amounts. After appropriate hybridization, the generated products were digested with the SURVEYOR Nuclease included in the kit. Then the digested products were visualized through gel electrophoresis (2.5% gel).

and the resultant vector was named as pYW19gRNA-BseRI (Figure 1a). In the vectors containing various promoters driving gRNA expression, a 20-nt protospacer sequence (5′-GATGCAGATGGAATGGTATC-3′) targeting on the C. beijerinckii phosphotransacetylase gene (pta) fused with the gRNA sequence was included. The first two promoter-screening vectors pYW19gRNA-Cbe0144p1 (with 284 bp RNA polymerase (Cbei_0144) promoter) and pYW19gRNA-sCbe2478p (with sRNA sCbei_2478 promoter) were constructed in the same manner as was the case for pYW19gRNA-BseRI, except that alternative gBlocks (gBLK02 for pYW19gRNA-Cbe0144p1 and gBLK03 for pYW19gRNA-sCbe2478p, see Table S2) were synthesized and used in place of gBLK01 as for pYW19gRNABseRI. The other vectors for screening of promoters were constructed based on pYW19gRNA-BseRI. First, PCR fragments containing respective promoter sequences were amplified using corresponding pairs of primers (P03 to P14, Table S2) and C. beijerinckii genomic DNA (gDNA) as template; then pYW19gRNA-BseRI was digested with BseRI and the respective PCR fragment was inserted into the vector through Gibson Assembly in order to obtain the corresponding vector. Vector pYW19-pta was constructed using Gibson Assembly as follows. The strong constitutive thiolase gene promoter (thlP) was selected for Cas9 expression, and was amplified using primers P17 and P18 from C. beijerinckii gDNA. The Cas9 open reading frame (ORF) was amplified from the plasmid pMJ806 (purchased from Addgene)11 using primers P19 and P20. A 784 bp gBlock (gBLK04, Table S2) was synthesized by IDT, including thlT, sCbei_5830 promoter for gRNA expression, the 20-nt guiding sequence (5′-GATGCAGATGGAATGGTATC-3′) targeting on pta fused with the chimeric gRNA sequence,8 and a transcription terminator derived from S. pyogenes.36 The three fragments cited above (thlP, Cas9 ORF and the gBLK04) were finally assembled and inserted into pTJ1 between the ApaI and NotI restriction enzyme sites, and the resultant vector was named as pYW19-pta. Additional detailed construction procedures for other vectors are included in Supporting Information. RNA Isolation, cDNA Synthesis, and Semiquantitative Reverse Transcription PCR (sqRT-PCR). To screen promoters for gRNA expression, vectors for gRNA expression driven by various promoters were transformed into C. beijerinckii 8052. Positive transformants were identified and grown in liquid TGY medium supplemented with 25 μg/mL of Erm. Cell pellets were harvested when OD600 reached ∼0.80 and total RNA were isolated using Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA) following the manufacturer’s protocol. On-column DNase treatment was conducted in order to get rid of DNA contamination. Then cDNA was synthesized from 1 μg total RNA using the Protoscript M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs Inc., Ipswich, MA) with a sequence specific primer P16 (annealing to the expected gRNA sequence, Table S2) for the synthesis. The cDNA product was diluted as appropriate and used as the template for PCR. PCR was carried out using Taq DNA polymerase (Promega, Madison, WI) with primers P15 and P16 (Table S2). Subcultivation Procedure for Isolating Desirable Mutants When the Lactose Inducible Promoter Was Used for Cas9 Expression. A similar protocol as described by Al-Hinai et al.2 with modification was used in this study to isolate the desirable mutants when the lactose inducible promoter was employed for Cas9 expression. After C. beijerinckii was transformed with appropriate plasmid and culture recovered following

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00060. Additional information including the detailed construction procedures for additional various vectors. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 1-217-333-8224. Fax: 1-217-244-2517. E-mail: blaschek@ illinois.edu. Present Address ∇

215 Tom E. Corley Building, Biosystems Engineering Department, Auburn University, Auburn, Alabama 36849, United States.

Author Contributions

Y.W., T.L., Y.S.J. and H.P.B. conceived the idea and planned the experiments. Y.W., Z.T.Z., S.O.S. and P.L. performed the experiment. Y.W., S.O.S., T.L., Y.S.J. and H.P.B. wrote the manuscript. J


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ACS Synthetic Biology Notes

(8) Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., and Church, G. M. (2013) RNA-guided human genome engineering via Cas9. Science 339 (6121), 823−826. (9) Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., and Zhang, F. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819−823. (10) DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., and Church, G. M. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41 (7), 4336−4343. (11) Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012) A programmable dual-RNA−guided DNA endonuclease in adaptive bacterial immunity. Science 337 (6096), 816− 821. (12) Jiang, W., Bikard, D., Cox, D., Zhang, F., and Marraffini, L. A. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31 (3), 233−239. (13) Oh, J. H., and van Pijkeren, J. P. (2014) CRISPR−Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res. 42 (17), e131. (14) Cobb, R. E., Wang, Y., and Zhao, H. (2014) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4 (6), 723−728. (15) Huang, H., Zheng, G., Jiang, W., Hu, H., and Lu, Y. (2015) Onestep high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim. Biophys. Sin. 47 (4), 231−243. (16) Tong, Y., Charusanti, P., Zhang, L., Weber, T., and Lee, S. Y. (2015) CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth. Biol. 4 (9), 1020−1029. (17) Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., and Yang, S. (2015) Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. Appl. Environ. Microbiol. 81 (7), 2506−2514. (18) Xu, T., Li, Y., Shi, Z., Hemme, C. L., Li, Y., Zhu, Y., Van Nostrand, J. D., He, Z., and Zhou, J. (2015) Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase. Appl. Environ. Microbiol. 81 (13), 4423−4431. (19) Pidot, S., Ishida, K., Cyrulies, M., and Hertweck, C. (2014) Discovery of clostrubin, an exceptional polyphenolic polyketide antibiotic from a strictly anaerobic bacterium. Angew. Chem., Int. Ed. 53 (30), 7856−7859. (20) Wang, Y., and Blaschek, H. P. (2011) Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii NCIMB 8052. Bioresour. Technol. 102 (21), 9985−9990. (21) Ezeji, T., and Blaschek, H. P. (2008) Fermentation of dried distillers’ grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia. Bioresour. Technol. 99 (12), 5232−5242. (22) Liao, C., Seo, S.-O., Celik, V., Liu, H., Kong, W., Wang, Y., Blaschek, H., Jin, Y.-S., and Lu, T. (2015) Integrated, systems metabolic picture of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum. Proc. Natl. Acad. Sci. U. S. A. 112 (27), 8505−8510. (23) Wang, Y., Zhang, Z.-T., Seo, S.-O., Choi, K., Lu, T., Jin, Y.-S., and Blaschek, H. P. (2015) Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J. Biotechnol. 200, 1−5. (24) Wang, Y., Li, X., Mao, Y., and Blaschek, H. (2011) Singlenucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq. BMC Genomics 12 (1), 479. (25) Wang, Y., Li, X., Mao, Y., and Blaschek, H. (2012) Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq. BMC Genomics 13, 102. (26) Ma, H., Wu, Y., Dang, Y., Choi, J.-G., Zhang, J., and Wu, H. (2014) Pol III promoters to express small RNAs: delineation of transcription initiation. Mol. Ther.–Nucleic Acids 3, e161. (27) Hartman, A. H., Liu, H., and Melville, S. B. (2011) Construction and characterization of a lactose-inducible promoter system for controlled gene expression in Clostridium perfringens. Appl. Environ. Microbiol. 77 (2), 471−478.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Department of Energy (DOE) grant #2011-01219 to HPB. We thank Dr. Terry Papoutsakis for providing the pKO_mazF plasmid. We also thank Mr. Wenyan Jiang (from Dr. Luciano A. Marraffini’s group at The Rockefeller University), Dr. Esteban Toro (from Dr. Adam P. Arkin’s group at UC-Berkeley), Dr. Jason Peters (from Dr. Carol Gross’ group at UC-San Francisco), and Dr. Martin Jinek (from Dr. Jennifer Doudna’s group at UC-Berkeley) for their helpful discussions. We thank Dr. Jie Zhang and Dr. Shaohua Wang (both from Dr. Yi Wang’s group at Auburn University) for insightful discussions and assistance with the figures. We thank Dr. Roderick I. Mackie for sharing his lab facilities.

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ABBREVIATIONS ack, acetate kinase gene; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; Cas, CRISPR-associated; cPCR, colony PCR; DSBs, double-strand breakages; Erm, erythromycin; gDNA, genomic DNA; gRNA, single chimeric guide RNA; HR, homologous recombination; NHEJ, nonhomologous end-joining; ORF, open reading frame; PAM, Protospacer Adjacent Motif; aPAM, artificial PAM; mPAM1, modified PAM1; pta, phosphotransacetylase gene; SNM, single nucleotide modification; Sp, spectinomycin; sqRT-PCR, semiquantitative reverse transcription PCR; sRNA, small RNA; TGY, tryptone-glucose-yeast extract; TGYE25, TGY plate supplemented with 25 μg/mL of Erm; TGYSp750, TGY plate supplemented with 750 μg/mL Sp; TGYLE25, TGY plate supplemented with 40 mM lactose and 25 μg/mL of Erm; TGYLSp750, TGY plate supplemented with 40 mM lactose and 750 μg/mL Sp; thlP, thiolase gene (Cbei_0411) promoter; thlT, thiolase gene (Cbei_0411) terminator; VIE, vector integration event; WT, wild type

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