How to make sure my amplification of the GeCKOv2 CRISPR/Cas9 library contains all plasmis

[liushih...@gmail.com]

Hi!

I followed the protocol for GeCKOv2 pooled library amplification to expand the library. And I want to make sure my amplification of the library contains all plasmids. I don't understand why the science paper provides the #2 PCR primer is barcodes? Can I use primer1 the paper provides to PCR and then use another PCR primer I designed :F-GTTTTAAAATGGACTATCATATGCTTACCG, R-GCCAAGCTTAAAAAAGCACCGACTCGGTGC to make the second PCR? After PCR use my designed primers, I sent my PCR product to sequence. Finally, the results was not successful. It only can detected 10% percent of the sgRNA in the library. How can I do? Can you give me some advice? Thank you!

[Neville Sanjana]

Sorry to hear about your troubles. Only detecting 10% of sgRNAs is definitely a problem. It is possible that your amplification had some issues. We have a detailed library amplification protocol here: 

http://genome-engineering.org/gecko/?page_id=15    (GeCKO Library v1 and v2 transformation protocol )

You don't mention which vector you are using but for lentiGuide-Puro (GeCKOv2 human or mouse library) or lentiCRISPRv1 (GeCKOv1 human library), you can directly use our Illumina primers listed here and do just a single PCR step:

http://genome-engineering.org/gecko/?page_id=15  (Readout primers for Illumina sequencing of sgRNA cassette )

If you're using lentiCRISPRv2 (GeCKOv2 human or mouse library), you need to first amplify using an adaptor primer (also given in the spreadsheet referenced above) in order to add a place for our Illumina primers to anneal. 

Of course, you can always design your own Illumina primers for exactly the vector you're using. If this is something you haven't done before, just take a look at the spreadsheet above which breaks down each element in the primer design.

Best wishes,

Neville

On Sat, Feb 27, 2016 at 7:20 AM, <liushih...@gmail.com> wrote:

Hi!

I followed the protocol for GeCKOv2 pooled library amplification to expand the library. And I want to make sure my amplification of the library contains all plasmids. I don't understand why the science paper provides the #2 PCR primer is barcodes? Can I use primer1 the paper provides to PCR and then use another PCR primer I designed :F-GTTTTAAAATGGACTATCATATGCTTACCG, R-GCCAAGCTTAAAAAAGCACCGACTCGGTGC to make the second PCR? After PCR use my designed primers, I sent my PCR product to sequence. Finally, the results was not successful. It only can detected 10% percent of the sgRNA in the library. How can I do? Can you give me some advice? Thank you!

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[liushih...@gmail.com]

Dear Neville

Thank you for your letter. I use Human GeCKO v2 Library (2 plamsid system-lentiGuide-Puro) and Mouse GeCKO v2 Library (2 plamsid system-lentiGuide-Puro). I want to know what's the meaning of this protocol I appended in the picture. I should collect all the bacteria from 20 10cm plates together to extract plasmid or collect bacteria from each 10cm plate separately? In addition, Can you give me the sequence of the plasmid of the GeCKO v2 library? Thank you very much.

在 2016年2月27日星期六 UTC+8下午12:03:54，Neville Sanjana写道：

[Neville Sanjana]

For the protocol, you should pool the bacteria from all plates together into one pellet. Please make sure to weigh the resulting bacterial pellet so that you can divide it into the right number of maxi preps (otherwise, using just a single maxi column will clog and fail).

Sequences are available for download here:

http://genome-engineering.org/gecko/?page_id=15  : GeCKO library indexes (sgRNA sequences and genes targeted)

Best wishes,

Neville

[Shihua Liu]

Dear Neville

Thank you for you letter. I want the sgRNA plasmid skeleton not the sgRNA sequence. I have got the sgRNA suquences already. What's the length of the first PCR product? At the same time, I don't understand why the illumina primers designed like that. I don't know the principle. I don't know what's the illumina seq, barcode seq and the stagger. Should I put 12 pairs PCR#2 primers together to PCR or each pair of primers separately?

在 2016年2月28日星期日 UTC+8上午12:36:52，Neville Sanjana写道：

[Shihua Liu]

Dear Neville

I have got the lentiGuide-Puro sequences.

在 2016年2月28日星期日 UTC+8上午12:36:52，Neville Sanjana写道：

For the protocol, you should pool the bacteria from all plates together into one pellet. Please make sure to weigh the resulting bacterial pellet so that you can divide it into the right number of maxi preps (otherwise, using just a single maxi column will clog and fail).

[Shihua Liu]

Dear Neville

Thank you for your letter. What's the length of the first PCR product? At the same time, I don't understand why the illumina primers designed like that. I don't know the principle. I don't know what's the illumina seq, barcode seq and the stagger. Should I put 12 pairs PCR#2 primers together to PCR or each pair of primers separately? 

在 2016年2月28日星期日 UTC+8上午12:36:52，Neville Sanjana写道：

For the protocol, you should pool the bacteria from all plates together into one pellet. Please make sure to weigh the resulting bacterial pellet so that you can divide it into the right number of maxi preps (otherwise, using just a single maxi column will clog and fail).

[Neville Sanjana]

Hi Shihua,

For lentiGuide-Puro, if you're using our PCR#1 & 2 primers, your PCR#1 product should be 317 bp. (See Science 2014 paper for PCR#1 primer sequences.)

For Illumina sequencing, our PCR#2 primers add on the required Illumina sequences along with barcodes. The stagger region is to avoid monotemplate, which can be an issues with low complexity sequencing (i.e the region before the guide sequence). If you haven't done Illumina sequencing before, it might be best to consult with others in your lab to get a more comprehensive understanding before starting the readout. The GeCKO readout is pretty much the same as any targeted amplicon sequencing library prep.

Best wishes,

Neville

[Neville Sanjana]

For lentiGuide-Puro, you can use the exact same PCR#1 and #2 primers as in the Science paper (or design your own).

We always gel purify but others have reported success with SPRI beads and other cleanup methods. I think the key is to eliminate any possible primer (or primer-dimer) contamination.

Best,

Neville

On Tue, Mar 8, 2016 at 10:17 AM, Shihua Liu <liushih...@gmail.com> wrote:

Dear Neville

The 2 steps PCR primers in the science 2014 paper is not the same as the primer in the nature 2014 paper "Improved vectors and genome-wide libraries for CRISPR screening" .Which primers should I use? And another question is whether I should purify the second PCR product by gel after the second PCR before send it to illumina sequence? Thank you for your answer.

Best wishes 

在 2016年2月29日星期一 UTC+8下午3:14:50，Neville Sanjana写道：

[Shihua Liu]

Dear Neville

I use the human GeCKO v2 library (2 plasmid system-lentiguide-puro). I still can't understand why I should use 12 different PCR#2 barcode primers to make the second PCR#2. And  should I have the 12 pairs of primers together to make the second PCR#2? Waiting for your answer. Thank you very much.

在 2016年3月9日星期三 UTC+8下午10:42:14，Neville Sanjana写道：

[Neville Sanjana]

Hi Shihua,

The 12 different F primers (not the 12 primers, since we only need a single-end forward read) are necessary to increase sequence diversity to avoid monotemplate issues during readout. Monotemplate is a common problem when doing targeted amplicon sequencing where the sequences have low diversity in specific regions, which is exactly the case here. Even with the stagger complexity, you might also need to spike in some PhiX for Illumina sequencing. 

If you haven't dealt with these issues before, I recommend speaking with your sequencing facility to find out more. Normally they will have good advice on how to prepare the libraries to avoid monotemplate.

Best,

Neville

[Shihua Liu]

Dear Neville

I can see 12 different R primers from the website http://genome-engineering.org/gecko/?page_id=15 Readout primers for Illumina sequencing of sgRNA cassette :the excel. So I still can't understand my question.

在 2016年3月14日星期一 UTC+8下午8:26:22，Neville Sanjana写道：

[Neville Sanjana]

Hi Shihua,

The 12 reverse primers can be used to barcode different biological samples. With barcoded samples, you can then multiplex them into a single sequencing run. Hope that makes sense.

Best,

Neville