Inferring gene function with Cas9 can be done by:

• gene knock out: in this case, by abrogating the function of a specific gene you can infere - based on the phenotype you observe - what the function of the gene can be. In this case, the limitations are two:
  • You have to ensure double alleles knock-out: if the gene is aplosufficient - which means that it can cover its function with just “ one copy of the gene “ - you won’t see any phenotype. To analyze this you can design a PCR-based experiment in witch you design primers to bind specifically to one of the two alleles and see if you get a PCR product in both cases or just one.
  • Off-target: one of the problem when it comes to Cas9 ( as well as in TALENs and ZFNs ) is that off-target may occur! Now the problem is > if you have off-target how can you say that the phenotype observed is due to the gene you just knock-out instead of the off-targets generated? A first analysis may come from a gene sequencing in which you can see where those off-targets are and see if they are relevant. If the off-target is in a non active region (an intron ), if it does not result in a frameshift of the reading frame, you may already say that the effect observed is due to your knock-out. Another experiment may be to knock in again the region you just cut and see if the effect observed is suppressed.
• Epigenetic modifications: nothing thrilles me more the possibility to actually drive epigenetic modifications at a specified target! Why? Cause it is reversible and does not affect directly the gene sequence, this means that if you want to create an epigenetic-based therapy ( p53 re-activation, CCR5 suppression ) you don-t have to modify the actual DNA of the patients but only the way it is expressed! Now, due to the fact that Epigenetics is complex and dynamic driving Epigenetic modifications at the desired locus is always tricky. Many groups are reporting effective way to deliver Epigenetic modifications, the group I’m working with as well ( Designer epigenome modifiers enable robust and sustained gene silencing in clinically relevant human cells. ) but still to me there some issues that still prevent Cas9 to be 100% reliable when it comes to interrogate Epigentics ( what follows are personal opinions based on what I have read so far on research literature and based on discussion during lab meetings, of course more experienced researchers will provide us with usefull insights)>
  • In most of the experiment done so far ( I will bring as example this paper that I recently read DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A ), only one or two Epigenetic factors ( like DNA methylation transferase 3A ) are took into account and they show that is possible to achieve a significant DNA methylation and DNA silencing BUT:
    • Unless you don’t stably transfect your cells, those Epigenetics changes are erased after a while ( Epigentics is dynamic and there are a lot of endogenous mechanisms that assure that correct epigenetic marks are deposited ). So, the lack of solid epigenetic mark delivery may make hard to infere the actual effect of this modification. Maybe you achieve the silencing of a gene, but the result of this - like some cascade effects - can-t be seen before a certain amount of time and maybe before you are able to see it the epigenetic modifications you induced have already been reverted.
    • The efficiency of Epigenetic modifications delivery ( how is clearly stated in the paper I provide you ) are strictly dependent on the target, some target are more sensible and responsive to the induced modifications while other not ( for example some targets are easily methylable while other don’t ( this may due to:
    • 1) difference in chromatin 3D structure: it makes more easier or not for the Cas9 to access the targeted point
    • 2) more than one factor needed: of course the whole Epigentics does not really just on the DNMT3A but on hundreds of different factors! So, fusing a Cas9 to just one factor per time may be not effective at all! What would be interesting to see, maybe taking advantage of this SunTag system, is to fuse Cas9 to different factors (combinatorial approach ) and see if a synergistic effect ( how I would expect due the complexity of Epigenetic mechanisms ) can be observed

However Cas9 , regardless the limitations that step by step will be for sure solved, offers a powerful and sharp tool for the interrogation of Gene function and in particular of complex gene regulatory mechanisms like Epigentics is!

I hope it helps!

Source : Antonio Carusillo, PhD Candidate in Genetic Engineering (Marie Curie) at University of Freiburg

Dear Wei...

Will you check this link:

Chapter CRISPR/Cas9: a versatile genome-editing tool for crop improvement

Also, Try to reach Dr.Yuan the author.

Best Luck

I wanna find limitations of mammal cell targeting in vivo and vitro.

Inferring gene function with Cas9 can be done by:

• gene knock out: in this case, by abrogating the function of a specific gene you can infere - based on the phenotype you observe - what the function of the gene can be. In this case, the limitations are two:
  • You have to ensure double alleles knock-out: if the gene is aplosufficient - which means that it can cover its function with just “ one copy of the gene “ - you won’t see any phenotype. To analyze this you can design a PCR-based experiment in witch you design primers to bind specifically to one of the two alleles and see if you get a PCR product in both cases or just one.
  • Off-target: one of the problem when it comes to Cas9 ( as well as in TALENs and ZFNs ) is that off-target may occur! Now the problem is > if you have off-target how can you say that the phenotype observed is due to the gene you just knock-out instead of the off-targets generated? A first analysis may come from a gene sequencing in which you can see where those off-targets are and see if they are relevant. If the off-target is in a non active region (an intron ), if it does not result in a frameshift of the reading frame, you may already say that the effect observed is due to your knock-out. Another experiment may be to knock in again the region you just cut and see if the effect observed is suppressed.
• Epigenetic modifications: nothing thrilles me more the possibility to actually drive epigenetic modifications at a specified target! Why? Cause it is reversible and does not affect directly the gene sequence, this means that if you want to create an epigenetic-based therapy ( p53 re-activation, CCR5 suppression ) you don-t have to modify the actual DNA of the patients but only the way it is expressed! Now, due to the fact that Epigenetics is complex and dynamic driving Epigenetic modifications at the desired locus is always tricky. Many groups are reporting effective way to deliver Epigenetic modifications, the group I’m working with as well ( Designer epigenome modifiers enable robust and sustained gene silencing in clinically relevant human cells. ) but still to me there some issues that still prevent Cas9 to be 100% reliable when it comes to interrogate Epigentics ( what follows are personal opinions based on what I have read so far on research literature and based on discussion during lab meetings, of course more experienced researchers will provide us with usefull insights)>
  • In most of the experiment done so far ( I will bring as example this paper that I recently read DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A ), only one or two Epigenetic factors ( like DNA methylation transferase 3A ) are took into account and they show that is possible to achieve a significant DNA methylation and DNA silencing BUT:
    • Unless you don’t stably transfect your cells, those Epigenetics changes are erased after a while ( Epigentics is dynamic and there are a lot of endogenous mechanisms that assure that correct epigenetic marks are deposited ). So, the lack of solid epigenetic mark delivery may make hard to infere the actual effect of this modification. Maybe you achieve the silencing of a gene, but the result of this - like some cascade effects - can-t be seen before a certain amount of time and maybe before you are able to see it the epigenetic modifications you induced have already been reverted.
    • The efficiency of Epigenetic modifications delivery ( how is clearly stated in the paper I provide you ) are strictly dependent on the target, some target are more sensible and responsive to the induced modifications while other not ( for example some targets are easily methylable while other don’t ( this may due to:
    • 1) difference in chromatin 3D structure: it makes more easier or not for the Cas9 to access the targeted point
    • 2) more than one factor needed: of course the whole Epigentics does not really just on the DNMT3A but on hundreds of different factors! So, fusing a Cas9 to just one factor per time may be not effective at all! What would be interesting to see, maybe taking advantage of this SunTag system, is to fuse Cas9 to different factors (combinatorial approach ) and see if a synergistic effect ( how I would expect due the complexity of Epigenetic mechanisms ) can be observed

However Cas9 , regardless the limitations that step by step will be for sure solved, offers a powerful and sharp tool for the interrogation of Gene function and in particular of complex gene regulatory mechanisms like Epigentics is!

I hope it helps!

Source : Antonio Carusillo, PhD Candidate in Genetic Engineering (Marie Curie) at University of Freiburg

• Unless you don’t stably transfect your cells, those Epigenetics changes are erased after a while

So how to stably transfect your cells？（improve the stability of plasmid or insert sgRNAs, dCas9-Suntag in Rosa26 loci).

By the way, do any people investigate what effectors cause epigenetic modification? I assume that epigenetic modification could result from inactivation of epigenetics-effector/protein or gene mutation in some loci unless why epigenetic modification is dynamic and the loci that have been changed revert into previous situation again.

One of its limitations of dCas9 is its toxicity. See one recent report (2018):

A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9

https://www.nature.com › nature communications › articles