Hi John,

We've used S. pyogenes Cas9 extensively in E. coli (with both crRNA and sgRNA targeting) and have also observed activity in Vibrio cholerae (with crRNA targeting only thus far). It's true that Cas9 has primarily been used in eukaryotes, but this is probably due to a lack of alternative methods relative to bacteria.

We have not yet observed Cas9 to completely fail in any species, though in the early days I heard from numerous labs that they observed toxicity or lack of activity in species where we did not, presumably due to differing expression levels. Given the diversity of species we've tested (E. coli, Vibrio, yeast, C. elegans, human cells) and the many others that have been published, I'd be somewhat surprised to see it reliably fail, though I haven't heard anyone test it in archaea.

In addition to S. pyogenes Cas9, we've also employed three additional Cas9 orthologs from N. meningitidis, S. thermophilus system #1, and T. denticola. N. meningitidus and S. thermophilus #1 also function in Vibrio cholerae (targeted with crRNA). Note that all of these have different PAM requirements for cutting. It's possible you might have more luck with one of them if there are other factors in your organism of interest that may be reacting specifically to the sequence of your guide RNA, which is unlikely but possible.

As for failure modes:

The activities of different spacer sequences can vary considerably and also interact nonlinearly with the PAM (which I define as the sequence of at least 8 bases 3' of the protospacer). See our Nature Methods paper on orthogonal Cas9 proteins for details on the alternative Cas9 proteins and our findings with respect to spacer activity. Cas9 systems using a high-activity spacer to target a plasmid with a viable PAM cannot coexist with that plasmid, even with selection. Even a low-activity spacer will still eliminate the plasmid from most cells over 24 hours without selection.

Any secondary structure arising from the interaction of 5' sequences prior to the 20 bp spacer (as either crRNA or sgRNA) with the spacer itself or the repeat/guide can interfere with activity, sometimes abrogating it entirely.

Overexpression of any Cas9 protein, even nuclease-null variants, appears to be toxic in all organisms we've worked with, possibly because it binds fairly tightly to PAMs even without a guide. However, Cas9 expressed at an appropriate level in E. coli incurs no significant fitness cost in our hands. Tuning expression is critical.

Above all, I strongly recommend targeting a plasmid rather than the chromosome for initial testing if at all possible. If your species of interest can't be transformed efficiently, conjugation seems to work fine - conjugate your Cas9 vector or a control into cells containing a targeted plasmid in the absence of selection, select for the new vector in liquid culture, and plate to detect differential survival of the target plasmid.

To summarize:

- tune the expression level of Cas9 to a nontoxic level for your organism

- target a plasmid for initial testing

- ensure transcription starts with your 20 bp spacer sequence

- check spacers to verify they don't form secondary structure with the guide/repeat

- try multiple spacers and PAMs if your first attempt fails

- different Cas9 orthologs might function better or worse in certain species

- it's possible that crRNA targeting may be more robust in some organisms that react poorly to extended sgRNA hairpins (though we have no hard data on this)

Kevin

Hi John,

We've used the double mutant for regulation in E. coli (a Cell paper last year) and we're recently working with Jamie Cate's group, for efficient manipulation of wilder yeasts. It has proven a little trickier to get it working efficiently in diverse bacteria... there seems to be some toxicity of the Cas9 protein in a lot of cases. Jennifer Doudna and Luciano Marrafini have used it for genome editing in E. coli and Streptococcus pneumoniae (constructs available from AddGene I think). I haven't yet seen it widely used in diverse bacteria (or Archaea however.

Adam

Dear John,

We tried quite a lot but so far we have not been able to get the Cas9 system working in Agrobacterium tumefaciens. To eliminate promoter and expression level might be an issue, we tested three different promoters expressing Cas9 but all failed to show target cleavage. We also tested at least two promoters and terminators to express the guide RNA. We tried two targets in the chromosome for cleaveage but no success. At the moment the only thing left to test is changing the terminators so that there little to no extension of the guide RNA scaffold which might influence the secondary structure of the gRNA and binding to Cas9.

In E. coli, however, we have obtained first indications of Cas9 targeted cleveage. Indicating that our Cas9 and guide RNA expression system are functional.

I speculate that Cas9 system in bacterial species where CRISPR loci not detected, therere are alternative strategies to eliminate phage infections and that guideRNA might be degraded due to its secondary structure recognition in these bacterial species. Similarly, these immune reactions in various bacterial speices could be incompatible. We still have a long way ahead of us to get things sorted. We would be interested in collaborations to explore these further.

Hi John,

We've used S. pyogenes Cas9 extensively in E. coli (with both crRNA and sgRNA targeting) and have also observed activity in Vibrio cholerae (with crRNA targeting only thus far). It's true that Cas9 has primarily been used in eukaryotes, but this is probably due to a lack of alternative methods relative to bacteria.

We have not yet observed Cas9 to completely fail in any species, though in the early days I heard from numerous labs that they observed toxicity or lack of activity in species where we did not, presumably due to differing expression levels. Given the diversity of species we've tested (E. coli, Vibrio, yeast, C. elegans, human cells) and the many others that have been published, I'd be somewhat surprised to see it reliably fail, though I haven't heard anyone test it in archaea.

In addition to S. pyogenes Cas9, we've also employed three additional Cas9 orthologs from N. meningitidis, S. thermophilus system #1, and T. denticola. N. meningitidus and S. thermophilus #1 also function in Vibrio cholerae (targeted with crRNA). Note that all of these have different PAM requirements for cutting. It's possible you might have more luck with one of them if there are other factors in your organism of interest that may be reacting specifically to the sequence of your guide RNA, which is unlikely but possible.

As for failure modes:

The activities of different spacer sequences can vary considerably and also interact nonlinearly with the PAM (which I define as the sequence of at least 8 bases 3' of the protospacer). See our Nature Methods paper on orthogonal Cas9 proteins for details on the alternative Cas9 proteins and our findings with respect to spacer activity. Cas9 systems using a high-activity spacer to target a plasmid with a viable PAM cannot coexist with that plasmid, even with selection. Even a low-activity spacer will still eliminate the plasmid from most cells over 24 hours without selection.

Any secondary structure arising from the interaction of 5' sequences prior to the 20 bp spacer (as either crRNA or sgRNA) with the spacer itself or the repeat/guide can interfere with activity, sometimes abrogating it entirely.

Overexpression of any Cas9 protein, even nuclease-null variants, appears to be toxic in all organisms we've worked with, possibly because it binds fairly tightly to PAMs even without a guide. However, Cas9 expressed at an appropriate level in E. coli incurs no significant fitness cost in our hands. Tuning expression is critical.

Above all, I strongly recommend targeting a plasmid rather than the chromosome for initial testing if at all possible. If your species of interest can't be transformed efficiently, conjugation seems to work fine - conjugate your Cas9 vector or a control into cells containing a targeted plasmid in the absence of selection, select for the new vector in liquid culture, and plate to detect differential survival of the target plasmid.

To summarize:

- tune the expression level of Cas9 to a nontoxic level for your organism

- target a plasmid for initial testing

- ensure transcription starts with your 20 bp spacer sequence

- check spacers to verify they don't form secondary structure with the guide/repeat

- try multiple spacers and PAMs if your first attempt fails

- different Cas9 orthologs might function better or worse in certain species

- it's possible that crRNA targeting may be more robust in some organisms that react poorly to extended sgRNA hairpins (though we have no hard data on this)

Kevin

Hi Kevin (and the rest),

I'm Jose former postdoc at irene Chen's lab. I have a general question, what is the advantage of CRISPR over other methods (i.e. lambda red) for editing E. coli's genome? I can't think of a specific application that couldn't be achieved using the existing tools for regular biotechnology purposes.

Also, do you think that CAS9 could be potentially used in other gram negative bacteria? I would love to know about other examples.

Jose

Hi Jose,

It's primarily useful as a negative selection against almost any sequence and thus works in tandem with lambda Red. I can attest that it works beautifully in several Vibrio species that we've tried. Considering that it seems to function robustly in vitro and in species from all the walks of life, I'd be surprised if there were many species in which it did not work.

Kevin

Dear all,

Is cas9 protein expressed constitutively in bacteria? I was reading some basic informations about CRISPR/Cas9 system recently. Since it is a defence system against phage, it should be always well prepared? Is there any paper shows cas9 expression level in bacteria?

I was thinking for the bacteria that already have a type ii CRISPR system, maybe just transfer an sg RNA is enough for genome editing? (If the endogenous cas9 has enough activity..... )

Hi,Just to add on, recently our lab has tried targeted gene suppression using CRISPR tool in mycobacterium.We used codon optimised cas9 in order to minimise its toxic effect.

please find below the link of our paper for any further reference,if required:

http://www.nature.com/ncomms/2015/150225/ncomms7267/full/ncomms7267.html

Hi Kevin and John and all. I did some work on CRISPR CAS9  and found that combination of gRNA with wild type or defective Cas9, with or without target region resulted in reduction in growth rates of bacteria. My initial hypothesis was to test gRNA delivery into E. coli using nanoparticle. However, when toxicity of gRNA:Cas9 become an issue, I understand the experiment did not work. Now I am writing this part of the work as my phd thesis and I do not know how to discuss this matter in discussion section. Can anyone advice what could lead to toxicity of gRNA:Cas9  to the host, in the absence of target region of gRNA?