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Building genome-wide mutant resources in slow-growing mycobacteria

(2015)
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Abstract
Translating the S. pyogenes CRISPR/Cas9 system to a mycobacterial genetic engineering approach did not appear to be as straightforward as it has been for many other pro- and eukaryotic organisms. Early attempts to disrupt the sapM gene in M. bovis BCG by Cas9-induced dsDNA breaks, followed by NHEJ repair, were not successful. Transformation of a M. smegmatis strain with constitutive Cas9 expression plasmids, containing either an active or a catalytically-inactivated Cas9 variant, did not yield any transformants. In contrast, the same gene under control of the acetamidase promoter yields a comparable amount of cfu compared to the empty vector control. We were able to confirm (full-length) Cas9 expression in these clones, although the protein appears to be rapidly cleaved to smaller molecular weight fragments upon induction. We conclude that constitutive Cas9 expression (at least when expressed from the strong GroEL promoter) is likely to be lethal in M. smegmatis (and M. bovis BCG) and that this toxicity does not appear to correlate with Cas9’s endonuclease activity. Interference by the endogenous CRISPR machinery is unlikely to be an issue here, since no CRISPRlike loci are present in the M. smegmatis mc2155 strain57. While Cas9-induced toxicity does not seem to be a problem in most species where the system has been successfully employed, a single publication has reported on this issue in the single-cell alga Chlamydomas reinhardtii58. They showed that whereas transient expression of the Cas9 protein resulted in clear modifications within the target spacer region, no stable Cas9-expressing transformants could be obtained, including cells transformed with completely dead Cas9 variants. The authors conclude that controlled Cas9 expression will be crucial to make the approach operational in this organism. Based on this report, on our data and on other (unpublished) rumors within the online CRISPR community, we believe that Cas9-overexpression lethality and fine-tuning of its expression is one of, if not the major bottleneck hindering its success in various other (bacterial) organisms. Other Cas9 orthologs with characterized PAM requirements59 could be explored, some of which may show reduced toxicity in mycobacteria. As for the mycobacterial setup, controlled expression of Cas9 can thus be achieved using an inducible (acetamidase) promoter. However, so far we have no information on whether the sgRNA is (functionally) transcribed from the trp-tRNA promoter. Recently, an in vivo T7 RNA polymerase system driving sgRNA transcription has been described in Plasmodium falciparum60. The major advantage here is that the precise 5’ transcription start site is known, an essential feature for the sgRNA’s structural functionality. As the T7 RNA polymerase has already proven to work in Mycobacterium species61, we are currently exploring the use of this RNA polymerase (under control of the acetamidase promoter) to drive both cas9 and sgRNA transcription. During the preparation of this manuscript, a method was published on the CRISPR interference (CRISPRi) technology in mycobacteria62. Similarly as in other organisms, the authors use a Mycobacterium codon-optimized, nuclease-dead Cas9 construct which is able to bind but not cleave its genomic DNA target (guided by a sgRNA). The complex then obstructs the RNA polymerase on the targeted gene and prevents it from passing over this region, resulting in transcriptional repression and a gene knock-down phenotype. Interestingly, the authors use a tetracycline-inducible promoter to drive both Cas9 and sgRNA transcription63. The Cas9 expression plasmid is integrated into the Mycobacterium genome, while the sgRNA cassette is introduced on a replicating plasmid. This way, the authors are able to conditionally knock-down mutiple genes at once in both fast- and slow-growing mycobacteria. They monitored growth after Cas9 induction and observed no in vitro growth defects. As in our study, no toxicity is thus observed when expressed conditionally. This is the first report on an easy-to-implement technology for the conditional knock-down of genes in mycobacteria and will undeniably impact mycobacterial genetic studies in the future. Furthermore, this study indicates that the Cas9-sgRNA complex can be functionally expressed in Mycobacterium species. However, the authors did not mention the ‘conventional’ Cas9 endonuclease system to create targeted gene disruptions and it remains to be seen whether this targeted dsDNA break technology can be functionally implemented in Mycobacterium species. The key to success of this approach will be the possibility to pick up cells with mutant loci from an overwhelming background of WT survivors, since continuous selection against the parental target sequence (as is normally the case with constitutive Cas9 expression) will not be possible.

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Citation

Please use this url to cite or link to this publication:

MLA
Vandewalle, Kristof. “Building Genome-wide Mutant Resources in Slow-growing Mycobacteria.” 2015 : n. pag. Print.
APA
Vandewalle, K. (2015). Building genome-wide mutant resources in slow-growing mycobacteria. Ghent University. Faculty of Sciences, Ghent, Belgium.
Chicago author-date
Vandewalle, Kristof. 2015. “Building Genome-wide Mutant Resources in Slow-growing Mycobacteria”. Ghent, Belgium: Ghent University. Faculty of Sciences.
Chicago author-date (all authors)
Vandewalle, Kristof. 2015. “Building Genome-wide Mutant Resources in Slow-growing Mycobacteria”. Ghent, Belgium: Ghent University. Faculty of Sciences.
Vancouver
1.
Vandewalle K. Building genome-wide mutant resources in slow-growing mycobacteria. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2015.
IEEE
[1]
K. Vandewalle, “Building genome-wide mutant resources in slow-growing mycobacteria,” Ghent University. Faculty of Sciences, Ghent, Belgium, 2015.
@phdthesis{5990250,
  abstract     = {{Translating the S. pyogenes CRISPR/Cas9 system to a mycobacterial genetic engineering approach did not appear to be as straightforward as it has been for many other pro- and eukaryotic organisms. Early attempts to disrupt the sapM gene in M. bovis BCG by Cas9-induced dsDNA breaks, followed by NHEJ repair, were not successful. Transformation of a M. smegmatis strain with constitutive Cas9 expression plasmids, containing either an active or a catalytically-inactivated Cas9 variant, did not yield any transformants. In contrast, the same gene under control of the acetamidase promoter yields a comparable amount of cfu compared to the empty vector control. We were able to confirm (full-length) Cas9 expression in these clones, although the protein appears to be rapidly cleaved to smaller molecular weight fragments upon induction. We conclude that constitutive Cas9 expression (at least when expressed from the strong GroEL promoter) is likely to be lethal in M. smegmatis (and M. bovis BCG) and that this toxicity does not appear to correlate with Cas9’s endonuclease activity. Interference by the endogenous CRISPR machinery is unlikely to be an issue here, since no CRISPRlike loci are present in the M. smegmatis mc2155 strain57. While Cas9-induced toxicity does not seem to be a problem in most species where the system has been successfully employed, a single publication has reported on this issue in the single-cell alga Chlamydomas reinhardtii58. They showed that whereas transient expression of the Cas9 protein resulted in clear modifications within the target spacer region, no stable Cas9-expressing transformants could be obtained, including cells transformed with completely dead Cas9 variants. The authors conclude that controlled Cas9 expression will be crucial to make the approach operational in this organism. Based on this report, on our data and on other (unpublished) rumors within the online CRISPR community, we believe that Cas9-overexpression lethality and fine-tuning of its expression is one of, if not the major bottleneck hindering its success in various other (bacterial) organisms. Other Cas9 orthologs with characterized PAM requirements59 could be explored, some of which may show reduced toxicity in mycobacteria. As for the mycobacterial setup, controlled expression of Cas9 can thus be achieved using an inducible (acetamidase) promoter. However, so far we have no information on whether the sgRNA is (functionally) transcribed from the trp-tRNA promoter. Recently, an in vivo T7 RNA polymerase system driving sgRNA transcription has been described in Plasmodium falciparum60. The major advantage here is that the precise 5’ transcription start site is known, an essential feature for the sgRNA’s structural functionality. As the T7 RNA polymerase has already proven to work in Mycobacterium species61, we are currently exploring the use of this RNA polymerase (under control of the acetamidase promoter) to drive both cas9 and sgRNA transcription. During the preparation of this manuscript, a method was published on the CRISPR interference (CRISPRi) technology in mycobacteria62. Similarly as in other organisms, the authors use a Mycobacterium codon-optimized, nuclease-dead Cas9 construct which is able to bind but not cleave its genomic DNA target (guided by a sgRNA). The complex then obstructs the RNA polymerase on the targeted gene and prevents it from passing over this region, resulting in transcriptional repression and a gene knock-down phenotype. Interestingly, the authors use a tetracycline-inducible promoter to drive both Cas9 and sgRNA transcription63. The Cas9 expression plasmid is integrated into the Mycobacterium genome, while the sgRNA cassette is introduced on a replicating plasmid. This way, the authors are able to conditionally knock-down mutiple genes at once in both fast- and slow-growing mycobacteria. They monitored growth after Cas9 induction and observed no in vitro growth defects. As in our study, no toxicity is thus observed when expressed conditionally. This is the first report on an easy-to-implement technology for the conditional knock-down of genes in mycobacteria and will undeniably impact mycobacterial genetic studies in the future. Furthermore, this study indicates that the Cas9-sgRNA complex can be functionally expressed in Mycobacterium species. However, the authors did not mention the ‘conventional’ Cas9 endonuclease system to create targeted gene disruptions and it remains to be seen whether this targeted dsDNA break technology can be functionally implemented in Mycobacterium species. The key to success of this approach will be the possibility to pick up cells with mutant loci from an overwhelming background of WT survivors, since continuous selection against the parental target sequence (as is normally the case with constitutive Cas9 expression) will not be possible.}},
  author       = {{Vandewalle, Kristof}},
  language     = {{eng}},
  pages        = {{XV, 322}},
  publisher    = {{Ghent University. Faculty of Sciences}},
  school       = {{Ghent University}},
  title        = {{Building genome-wide mutant resources in slow-growing mycobacteria}},
  year         = {{2015}},
}