CRISPR gRNA library service

Pooled CRISPR guide RNA libraries, or gRNA libraries, are ideal for high-throughput screening of important molecular targets. These libraries leverage the efficiency and specificity of the CRISPR gene editing technology to either knock-out gene expression or transcriptionally activate genes in the genome.

GenScript offers a variety of gRNA library options, all of which contain sequences pre-validated by the Broad Institute, including (1) GeCKO libraries for genome-wide knockout of human and mouse genes, (2) CRISPR Synergistic Activating Mediator (SAM) libraries for transcriptional activation of every gene in the human and mouse genome, and (3) Pathway-focused gRNA libraries that genes with a specific function.

How are CRISPR libraries used?

Screening using the CRISPR technology is particularly advantageous because of its simplicity, specificity and versatility. The genome-wide GeCKO and SAM libraries were designed to target every gene in the mouse or human genes and knock-out or transcriptionally activate each gene, respectively. Pathway-focused gRNA libraries were designed from targets identified through the Drug Gene Interaction Database, providing application-focused gRNA libraries for more simplified screening. Libraries are amplified, packaged and transduced into host cells to generate mutant cell lines (Figure 1). The mutants can then be screened under specific conditions, such as by positive or negative selection, to identify important genes in a pathway.

Figure 1: How to use CRISPR gRNA libraries

 

Knockout Libraries

GeCKO library for genome-scale knock-out

The genome-scale CRISPR knock-out (GeCKO) v2 libraries enable rapid screening for loss-of-function mutations that produce a desired phenotype in human or mouse cells. GenScript is pleased to offer all eight GeCKO v2 CRISPR libraries developed in Feng Zhang's laboratory through a license with the Broad Institute.* GeCKO libraries are mixed pools of guide RNA targeting each gene and miRNA within the mouse or human genome; the improved GeCKO v2.0 libraries also contain non-targeting gRNA to serve as controls and utilize improved lentiviral vectors for high-titer lentivirus production and highly efficient transduction of primary cells. GeCKO libraries have been used in primary mouse and human cells, stem cells, cancer cells, and stable cell lines. GeCKO library screens allows you to identify genes that are essential for cell viability under different conditions, as well as genes whose loss enables drug resistance in cancer cells. For complete details of GeCKOv2 library design & construction, please see: Sanjana N.E., Shalem O., Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.

GeCKO v2 libraries are available as two half-libraries, A and B. Each library contains 3gRNAs per gene. Both libraries contain 1,000 non-targeting control sgRNAs. The A library contains gRNAs targeting miRNAs (4 sgRNAs per miRNA) as well. The A and B libraries can be purchased and delivered together to ensure complete representation; however, as more cells are required for both libraries, screening with library A is suggested.

Library	Sequence information
Human GeCKO Library A	65,383 sequences: ·         3 sgRNA targeting each of 19,050 genes; ·         4 sgRNA targeting each of 1,864 miRNA ·         1,000 control (non-targeting) sgRNAs
Human GeCKO Library B	58,028 sequences: ·         3 sgRNA targeting each of 19,050 genes; ·         1,000 control (non-targeting) sgRNAs
Mouse GeCKO Library A	67,405 sequences: ·         3 sgRNA targeting each of 20,611 genes; ·         4 sgRNA targeting each of 1,175 miRNA ·         1,000 control (non-targeting) sgRNAs
Mouse GeCKO Library B	62,804 sequences: ·         3 sgRNA targeting each of 20,611 genes; ·         1,000 control (non-targeting) sgRNAs

The gRNA libraries at GenScript are Industrial Grade and in-stock for immediate delivery (please allow 5-7 days for shipping) unless stated otherwise. Additional quantity options are available, but may require additional turnaround time. Email us for more information on additional library options.

Catalog #	Library Name	Vectors	Amount
SC1771-25 SC1771-100 SC1771-200	Human GeCKO Library A	pLentiCRISPR v2	25 µg 100 µg 200 µg
SC1774-25 SC1774-100 SC1774-200		pLentiGuide-Puro Accompanied by: pLentiCas9-Blast	25 µg 100 µg 200 µg
SC1777-25 SC1777-100 SC1777-200	Human GeCKO Library B	pLentiCRISPR v2	25 µg 100 µg 200 µg
SC1780-25 SC1780-100 SC1780-200		pLentiGuide-Puro Accompanied by: pLentiCas9-Blast	25 µg 100 µg 200 µg
SC1783-25 SC1783-100 SC1783-200	Mouse GeCKO Library A	pLentiCRISPR v2	25 µg 100 µg 200 µg
SC1843-25 SC1843-100 SC1843-200		pLentiGuide-Puro Accompanied by: pLentiCas9-Blast	25 µg 100 µg 200 µg
SC1849-25 SC1849-100 SC1849-200	Mouse GeCKO Library B	pLentiGuide-Puro Accompanied by: pLentiCas9-Blast	25 µg 100 µg 200 µg

GeCKO Library Deliverables

·  Pooled library (Industrial Grade) in 25 µg aliquots. The 100 µg and 200 µg library are delivered as 4 x 25 µg or 8 x 25 µg, respectively. 

   The libraries are delivered with a technical guide, MSDS and COA.

·  Mouse GeCKO Library A (dual vector): 100 µg pooled library (Research Grade)

· Scale up to 900 µg total, in increments of 100 µg; Industrial Grade available instead of Research Grade

· Final project report, including ABI files from Sanger sequencing for small-scale tests and statistical summary from next-generation sequencing validation. Complete raw NGS data available upon request

How to use GeCKO libraries

GeCKO libraries are a mixed pool of CRISPR guide RNAs that target every gene and miRNA in the genome. Each gRNA is cloned into a lentiviral vector optimized to produce high-titer virus for efficient lentiviral transduction of primary cells or cultured cell lines. 

Transcriptional Activation Libraries

CRISPR transcriptional activation SAM library

SAM libraries induce robust transcriptional activation for genome-wide gain-of-function screening

The SAM library enables gain-of-function screening in human cells by inducing robust, specific transcriptional activation of each coding region in the genome in a single screen. CRISPR/Cas9 Synergistic Activation Mediator (SAM) is a protein complex engineered to enable robust transcriptional activation of endogenous genes. SAM takes advantage of the specificity and ease of reprogramming of Cas9 nucleases, which are targeted to a specific locus in the genome by guide RNA. The SAM complex includes three transcription activation domains VP64, P65, and HSF1 which synergistically enhance transcription when targeted to any site within 200bp upstream of a transcription start site. SAM uses a nucleolytically inactive Cas9 to ensure that no strand breaks are introduced into endogenous genome.

The human genome-wide SAM library activates all known coding isoforms in the RefSeq database (23,430 isoforms) for gain-of-function screening. SAM libraries have been used in primary mouse and human cells, stem cells, cancer cells, and stable cell lines. SAM library screens allow you to identify genes that are essential for cell viability under different conditions, as well as genes whose loss enables drug resistance in cancer cells.

For complete details of SAM library design & construction, please see: Konermann S*, Brigham MD*, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O & Zhang F. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, doi:10.1038/nature14136.

SAM Library Contents

·         70,290 gRNA sequences; 3 sgRNA targeting each of 23,430 unique coding isoforms in the human genome.

·         Human SAM gRNA library is cloned into pLenti sgRNA(MS2)_zeo vector backbone.

·         Library is accompanied by the pLenti dCas-VP64_Blast and pLenti MS2-P-HSF1_Hygro plasmids which must be co-delivered.

SAM Library Deliverables

All SAM libraries are Industrial Grade and in-stock for immediate delivery (please allow 5-7 days for shipping). Additional quantity options are available, but may require additional turnaround time. Email us for more information on additional library options.

 

Catalog #	Library Name	Vectors	Amount
SC1770-25 SC1770-100 SC1770-200	Human SAM Library	pLenti sgRNA(MS2)_zeo; pLenti dCas9-VP64_Blast; pLenti MS2-P65-HSF1-Hygro.	25 µg 100 µg 200 µg
SC1754-25 SC1754-100 SC1754-200		pLenti sgRNA(MS2)_puro; pLenti dCas9-VP64_Blast; pLenti MS2-P65-HSF1-Hygro.	25 µg 100 µg 200 µg
SC1844-25 SC1844-100 SC1844-200	Mouse SAM Library	pLenti sgRNA(MS2)_puro pLenti dCas9-VP64_Blast; pLenti MS2-P65-HSF1-Hygro.	25 µg 100 µg 200 µg

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How to use SAM libraries for gain-of-function screens

The SAM library is a mixed pool of CRISPR guide RNAs that target immediately upstream of every transcription start site in the genome. For SAM gain-of-function screening, the human genome-wide SAM sgRNA library has to be combined with two additional SAM constructs – dCas9-VP64 and MS2-P-HSF1.

SAM Library Components

The SAM complex consists of three components:

 ·  A nucleolytically inactive Cas9-VP64 fusion

 ·  An sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop

 ·   The MS2-P65-HSF1 activation helper protein

    The inSC17xx-1corporation of three distinct activation domains - VP64, p65 and HSF1 – into the complex aids robust transcriptional activation through synergy. Each of the three components are encoded on a separate lentiviral vector as described below. All three vectors must be co-transduced in order for SAM to be active All SAM human genome-wide guide RNA library sequences are available in this .csv file.

 

Pathway-specific Libraries

 

Pathway-focused CRISPR gRNA library

Pathway-focused pooled gRNA libraries are ideal for targeted screening of specific pathways of importance. Designed using gene targets identified through the Drug Gene Interaction Database, these libraries are ideal for thorough screening of pathway or disease-focused genes. All gRNA sequences have been pre-designed and validated by the Broad Institute and cloned into pLentiCRISPR v2 vector backbones. All libraries are delivered Industrial Grade and ready for immediate lentiviral packaging. For more information on the gRNA sequences covered in the library, click on the library hyperlink.

The pathway-focused libraries are available as Industrial Grade, and available for next-day shipping (depending on availability).

 

Pathway	Gene #	gRNA #	Quantity	Amount
SC1822-25 SC1822-100	ABC Transporter	100	298	25 µg 100 µg
SC1823-25 SC1823-100	Adult Stem Cells	58	174	25 µg 100 µg
SC1824-25 SC1824-100	Angiogenesis	92	276	25 µg 100 µg
SC1825-25 SC1825-100	Apoptosis	68	204	25 µg 100 µg
SC1826-25 SC1826-100	cAMP & calcium Signaling Pathway	91	271	25 µg 100 µg
SC1827-25 SC1827-100	Cell Cycle	84	252	25 µg 100 µg
SC1828-25 SC1828-100	Cytochrome P450	57	171	25 µg 100 µg
SC1829-25 SC1829-100	Drug Resistance	348	1044	25 µg 100 µg
SC1830-25 SC1830-100	GPCR	280	840	25 µg 100 µg
SC1834-25 SC1834-100	Hormone Activity	108	324	25 µg 100 µg
SC1836-25 SC1836-100	Ion Channels (Potassium)	67	201	25 µg 100 µg
SC1837-25 SC1837-100	Tumor Metastasis	60	180	25 µg 100 µg

 

In addition to the in-stock libraries above, the following pathway-focused libraries are also available. Please allow 3-4 weeks for delivery. 

Click on the hyperlink to see the full gene list.

 

Pathway	Gene #	gRNA #
Acute myeloid leukemia	45	135
Adipogenesis	126	378
Alzheimer's disease	94	281
B cell receptor signaling pathway	58	174
Bladder cancer	37	111
Cardiovascular Disease	91	273
Chemokine signaling pathway	134	402
Chronic myeloid leukemia	57	171
Colorectal cancer	54	162
Diabetes	44	132
Drug Metabolism	34	102
Drug transporters	84	252
ES Cell Differentiation	334	1002
Extracellular Matrix & Adhesion	84	252
Growth Factor	161	483
Histone Modification	259	776
Nuclear Hormone Receptors	117	351
Oncogenes	289	867
Tumor Suppressor	720	2155
WNT Pathway	92	276

*Starting price for 25 μg pooled library. 100 μg quantity available upon request.

CRISPR gRNA library delivery specifications

·         Libraries are provided in 25 µg aliquots. Guide RNA sequences are cloned into pLentiCRISPR v2 vectors and delivered as Industrial Grade.

·         All libraries will be delivered with a technical guide, MSDS and COA.