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CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Library 

About:

 

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a microbial nuclease system involved in defense against invading phages. This system has been developed for efficient and precise genome editing in diverse organims. There are two distinct components to this system: (1) a guide RNA (gRNA) and (2) an endonuclease: the CRISPR associated (Cas) nuclease, Cas9. The gRNA/Cas9 complex is recruited to a specific genomic target sequence, which is immediately 5´ to a Protospacer Adjacent Motif (PAM) sequence. This recruitment of the gRNA/Cas9 complex to the genomic target sequence leads to Double Strand Break (DSB) in the DNA. This DSB can be repaired through one of two general repair pathways: (1) the Non-Homologous End Joining (NHEJ) DNA repair pathway or (2) the Homology Directed Repair (HDR) pathway. The NHEJ repair pathway often results in inserts/deletions (InDels) at the DSB site that can lead to frame shifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the targeted gene.

 

Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase (Cas9n) cuts only one strand of the target DNA, creating a single-strand break or ‘nick’. The Cas9 nickase is still able to bind DNA based on gRNA specificity and creates a single-strand break, which is repaired through the HDR pathway, using the intact complementary DNA strand as the template.

 

Cas9 with mutations in both catalyzing domains, also known as nuclease deficient Cas9 or dCas9, is able to bind DNA based on gRNA specificity but cannot create and nick or cut in the DNA. Nonetheless, this can be exploited to repress, activate or tag genes.

 

The Functional Genomics Facility at University of Colorado has received permission from the Zhang lab to distribute the plasmid reagents required for individual and genome-wide knock-out and activation of gene/genes using CRISPR technology. We hope to motivate and assist in adoption of CRISPR technology across labs in Colorado.

Zhang Lab CRISPR Pools

Pooled Libraries

Human GeCKO v2 Library in 1-Plasmid System (lentiCRISPRv2) (Addgene Cat# 1000000048).

(Vial A or B at 200ul @500ng/ul each)

 

Human GeCKO v2 Library in 2-Plasmid System (lentiGuide-Puro) (Addgene Cat# 1000000049).

(Vial A or B at 200ul @500ng/ul each + Glycerol Stock of lentiCas9-Blast)

 

Mouse GeCKO v2 Library in 1 Plasmid System (lentiCRISPRv2) (Addgene Cat# 1000000052).

(Vial A or B at 200ul @500ng/ul each).

Addgene: geckov2

 

Human Pooled SAM Library in 3 plasmid system (Addgene Cat# 1000000057). (Vial of SAM library 200ul @400 ng/ul and glycerol stocks of dCas9-VP64 and MS2-P65-HSF1 plasmids (Addgene #61426 & #61425).

Addgene: sam

Sequencing Primers

hU6-F (5'-GAGGGCCTATTTCCCATGATT-3')

 

or pLKO.1 (5'- GACTATCATATGCTTACCGT-3')

Custom Cloning for individual guide RNA

The Functional Genomics Facility has a streamlined process that is able to generate CRISPR constructs quickly, efficiently, and economically.

 

Generate plasmids to knockout genes into...

 

      1. All-in-one lentiCRISPRv2-puro

      2. All-in-one PX458/PX459

      3. Two-vector System lentiGUIDE-puro

 

 

Generate plasmids to activate genes into...

 

      1. lenti sgRNA(MS2)-puro/zeo

      2. sgRNA(MS2)

 

Custom cloning service = See "Product Prices" page

Turnaround = 2-3 weeks

For all other CRISPR cloning services, contact us.

References

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Science. 2013 Dec 12. (Article).

 

Multiplex Genome Engineering Using CRISPR/Cas Systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science. 2013 Jan 3 (Article).

 

Genome engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. (Article).

 

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Konermann S*, Brigham MD*, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O & Zhang F. Nature doi: 10.1038/nature14136 (2014) (Article).

 

Addgene CRISPR Guide

 

Addgene CRISPR Protocols

For technical support, FAQ, vector descriptions and maps, please visit the CRISPR section on our Resources page.

Arrayed CRISPR Library for human genes 

The Functional Genomics Facility has partnered with Sigma to bring you the first Arrayed CRISPR library. Knock out human genes without having to clone individual gRNA! You can search for the clone ID for your gene of interest in the Arrayed CRISPR Library Look-Up Sheet. To order CRISPR gRNAs, enter the clone IDs for your genes of interest into the appropriate section of the Order Form.

 

To learn more, contact us.

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