Target gene-CRISPR transcription activation

[selene...@gmail.com]

Hi everyone, I am new on CRISPR strategies and I am looking for a single gene transcriptional activation method. I was wondering if I can use the lentiSAM v2. plasmid (75112) for a single target gene activation and how to use it in a two-vector strategy.
Also I was questioning whether the sgRNAs design (flanking sequences) is the same for lentiSAMv2 backbone and lentisgRNA(MS2)_zeo (61427).

Selene

[selene...@gmail.com]

Is Synergistic Activation mediator intended solely for activation libraries or also for single gene targeting? 
I am thinking also about LentiCRISPRv2 and LentiGUIDE-Puro?

Len

Puro:

[selene...@gmail.com]

Basically, I have a plasmid dCAS9-VP64-MSloops (Addgene #75112) and I would like to know how to digest it to ligate my designed oligos. I was thinking to use BsmBI, but I am not sure about the digestion I will obtain

Il giorno martedì 14 novembre 2017 11:36:27 UTC+1, selene...@gmail.com ha scritto:

[Julia Joung]

Hi,

You can use golden gate assembly to very efficiently clone your sgRNA. Please refer to the validation section of our screening protocol for details on reaction setup.

Best,

Julia

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[Hiroyuki Kato]

Hi, 

I'm also planning to do CRISPRa targeting single gene (not genome wide screen).

1. It seems all the plasmid I need is lentiSAM v2 (75112) and lenti MPH v2 (89308), correct? (2-vector system)

2. LentiSAM v2 (75112) seems to be able to clone sgRNA, but I couldn't find specific protocol for this.

Is the following SAM cloning protocol also valid for LentiSAM v2?

http://sam.genome-engineering.org/static/SAM%20sgRNA%20spacer%20cloning%20protocol.pdf

If so do the golden gate reaction still necessary for cloning single sgRNA?

Or is it still possible using ordinary cloning protocol like in lentiCRISPR v2?

http://genome-engineering.org/gecko/wp-content/uploads/2013/12/lentiCRISPRv2-and-lentiGuide-oligo-cloning-protocol.pdf

Thanks in advance.

Best,

Hiroyuki

2017年11月25日土曜日 12時51分06秒 UTC+9 Julia Joung:

Hi,

You can use golden gate assembly to very efficiently clone your sgRNA. Please refer to the validation section of our screening protocol for details on reaction setup.

Best,

Julia

On Mon, Nov 20, 2017 at 11:32 AM, <selen...@gmail.com> wrote:

Basically, I have a plasmid dCAS9-VP64-MSloops (Addgene #75112) and I would like to know how to digest it to ligate my designed oligos. I was thinking to use BsmBI, but I am not sure about the digestion I will obtain

Il giorno martedì 14 novembre 2017 11:36:27 UTC+1, selene...@gmail.com ha scritto:

Hi everyone, I am new on CRISPR strategies and I am looking for a single gene transcriptional activation method. I was wondering if I can use the lentiSAM v2. plasmid (75112) for a single target gene activation and how to use it in a two-vector strategy.
Also I was questioning whether the sgRNAs design (flanking sequences) is the same for lentiSAMv2 backbone and lentisgRNA(MS2)_zeo (61427).

Selene

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[Julia Joung]

Hi Hiroyuki,

To answer your questions:

1. Yes those plasmids are sufficient.

2. Yes you can use the SAM cloning protocol, which uses Golden Gate assembly. Doing Golden Gate assembly will be much more efficient than the ordinary protocol, but both strategies work.

Best,

Julia

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[Ítalo Acuña]

One question, Will genetic engineering used for aesthetic reason? Such as having blue eyes, be taller, etc? Will that be allowed? If the answer is no, why? People Will want it for aesthetic reason for their children. 

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