Custom CRISPR sgRNA Pooled Libraries
In addition to our ready-to-use, off-the-shelf Genome-Wide CRISPR libraries, Cellecta also can generate Custom CRISPR sgRNA Pooled Libraries in as little as 6 weeks.
- Custom sgRNA libraries for CRISPR knockout, CRISPRa, and CRISPRi
- Get any guide and tracrRNA sequences desired–standard, HEAT, 10X capture sequences, etc.
- Cellecta has over 15 years experience making high quality pooled shRNA, barcode, and sgRNA libraries
As shown in the 2014 Science publications of Wang T, et al., and Shalem et al.; pooled libraries of CRISPR sgRNA-expressing constructs can be used to screen thousands of genes throughout a cell population and identify those that are required for a phenotype of interest such as viability, pathway activation, or the appearance of cell surface markers.
Drawing on our experience of over 15 years working with pooled shRNA, barcode, and sgRNA libraries, Cellecta has the expertise and capability to generate high-quality, complex CRISPR sgRNA libraries targeting virtually any defined sequences.
Screens have been done with variations of the CRISPR system with modified Cas9 proteins that alter the expression level of target genes. Screens can be run where gene targets are selectively inhibited (CRISPRi) or activated (CRISPRa) to assess the effect on phenotypes of interest. Cellecta will also design and provide custom libraries for CRISPRi and CRISPRa screens.
Finally, specialized applications often require sgRNA libraries with specialized designs. For example:
- Single cell applications may use guides with a modified tracrRNA containing specific sequences (e.g. capture sequences compatible with 10X Chromium Single Cell 3′ v3 Gel Beads)
- CRISPRa SAM system requires an additional stem loop in the sgRNA,
- Studies that combine RNA-Seq analysis with CRISPR screens can require an sgRNA cassette that is expressed as part of a transcript barcode,
- Other types of screens may need to have barcode or unique molecular identifier (UMI) sequences combined with sgRNAs.
Cellecta’s custom sgRNA construct and library service easily accommodates all the above variations and more. Just let us know what you need, and we will provide it.
Cellecta Custom CRISPR Libraries
1. Oligo Design and Synthesis
For sgRNAs targeting standard human and mouse protein-coding genes, you need only provide us with your list of targets. We design oligonucleotides encoding sgRNAs to your target genes based on the latest guidelines from published literature (e.g. Doench, et al., Nature 2016) as well as some other features we have optimized based on our screening work, to maximize effectiveness and minimize off-target activity. For more specialized applications, researchers may also provide their own guide sequences, or we can work with you to design specialized sgRNAs to meet your needs. In addition, we typically employ our improved sgRNA scaffold structure which incorporates the HEAT modifications unless other designs are preferred.
After the design step, we synthesize and clone the pool of oligos in any of our standard library vectors (See Resources tab, below) or, in many cases, we can make the library in a customer-provided vector. We can also include a panel of non-targeting, intron-targeting, non-specific cutting, and lethal sgRNA controls to provide reference standards for use when analyzing screening results.
3. Quality Analysis
Once the library is made, we isolate a few dozen constructs for full-insert Sanger sequencing to confirm the configuration of the sgRNA expression cassette and ensure correct insertion. We also deeply sequence all guide sequences by NGS, to confirm full representation of the oligo pool, and assess distribution. Libraries that do not meet our standards are remade.
On completion, we provide 500 µg of the plasmid library with:
- all sequence information on the sgRNA guides and vector
- the cloning site design
- primer information for sequencing
- NGS sgRNA distribution data
The whole process takes approximately two months once the gene list is finalized.
Optional Packaging: We also provide optional packaging services for lentiviral-based libraries so you can receive VSV-g pseudotyped viral particles ready to introduce onto cells for a screen.
Please email email@example.com to obtain a quotation.
- CRISPR Pooled Lentiviral sgRNA Libraries (Online Manual or PDF)
Custom sgRNA Library Vectors
The following CRISPR sgRNA cloning vectors are our standard custom library vectors. If interested in a vector not listed, please see the Vector Information page.
|Constitutive Library sgRNA Knockout Vectors||NGS Cassette*||Sequence**||Map|
|Constitutive Library sgRNA Knockout Vectors||NGS Cassette*||Sequence**||Map|
|Inducible Library sgRNA Knockout Vectors||NGS Cassette*||Sequence**||Map||Example Construct||NGS Amplicon|
Safety Data Sheets (SDS)
Next-Gen Sequencing of Samples from Genetic Screens
Cellecta CRISPR libraries are provided with a complete protocol and all sequencing information to enable researchers to perform high throughput genetics screens and analysis. However, we do also provide Next-Gen Sequencing (NGS) and analysis services for researchers running their own screens with our libraries. Just provide harvested cells for each time point or treatment condition (one sample), and we extract DNA, amplify, sequence, and assemble the data with some basic analysis.
You provide Cellecta with frozen cell pellets or tissue after screening…
…and Cellecta does the rest:
- Extracts genomic DNA
- Amplifies sgRNA sequences
- Performs NGS on the Illumina NextSeq or HiSeq
- Enumerates sgRNA counts from raw sequencing data
Please see the Next-Gen Sequencing and Analysis web page for additional information and how to order.
Custom CRISPR sgRNA Libraries
- Wang H, Lu B, Castillo J, Zhang Y, Yang Z, McAllister G, Lindeman A, Reece-Hoyes J, Tallarico J, Russ C, Hoffman G, Xu W, Schirle M, Cong F. Tankyrase Inhibitor Sensitizes Lung Cancer Cells to Endothelial Growth Factor Receptor (EGFR) Inhibition via Stabilizing Angiomotins and Inhibiting YAP Signaling. J Biol Chem. 2016 Jul 15;291(29):15256-66. doi: 10.1074/jbc.M116.722967. Epub 2016 May 26. PubMed PMID: 27231341.
- DeJesus R, Moretti F, McAllister G, Wang Z, Bergman P, Liu S, Frias E, Alford J, Reece-Hoyes JS, Lindeman A, Kelliher J, Russ C, Knehr J, Carbone W, Beibel M, Roma G, Ng A, Tallarico JA, Porter JA, Xavier RJ, Mickanin C, Murphy LO, Hoffman GR, Nyfeler B. Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62. Elife. 2016 Jun 28;5. pii: e17290. doi: 10.7554/eLife.17290. PubMed PMID: 27351204.
Custom shRNA Libraries
- Carugo A, Genovese G, Seth S, Nezi L, Rose JL, Bossi D, Cicalese A, Shah PK, Viale A, Pettazzoni PF, Akdemir KC, Bristow CA, Robinson FS, Tepper J, Sanchez N, Gupta S, Estecio MR, Giuliani V, Dellino GI, Riva L, Yao W, Di Francesco ME, Green T, D’Alesio C, Corti D, Kang Y, Jones P, Wang H, Fleming JB, Maitra A, Pelicci PG, Chin L, DePinho RA, Lanfrancone L, Heffernan TP, Draetta GF. In Vivo Functional Platform Targeting Patient-Derived Xenografts Identifies WDR5-Myc Association as a Critical Determinant of Pancreatic Cancer. Cell Rep. 2016 Jun 16. pii: S2211-1247(16)30661-1. doi: 10.1016/j.celrep.2016.05.063. [Epub ahead of print] PubMed PMID: 27320920.
- Munoz DM, Cassiani PJ, Li L, Billy E, Korn JM, Jones MD, Golji J, Ruddy DA, Yu K, McAllister G, DeWeck A, Abramowski D, Wan J, Shirley MD, Neshat SY, Rakiec D, de Beaumont R, Weber O, Kauffmann A, McDonald ER, Keen N, Hofmann F, Sellers WR, Schmelzle T, Stegmeier F, Schlabach MR. CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions. Cancer Discov. 2016 Jun 3. pii: CD-16-0178. [Epub ahead of print] PubMed PMID: 27260157.
- Mounir Z, Korn JM, Westerling T, Lin F, Kirby CA, Schirle M, McAllister G, Hoffman G, Ramadan N, Hartung A, Feng Y, Kipp DR, Quinn C, Fodor M, Baird J, Schoumacher M, Meyer R, Deeds J, Buchwalter G, Stams T, Keen N, Sellers WR, Brown M, Pagliarini RA. ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the androgen receptor. Elife. 2016 May 16;5. pii: e13964. doi: 10.7554/eLife.13964. [Epub ahead of print] PubMed PMID: 27183006.
- Bossi D, Cicalese A, Dellino GI, Luzi L, Riva L, D’Alesio C, Diaferia GR, Carugo A, Cavallaro E, Piccioni R, Barberis M, Mazzarol G, Testori A, Punzi S, Pallavicini I, Tosti G, Giacó L, Melloni G, Heffernan TP, Natoli G, Draetta GF, Minucci S, Pelicci P, Lanfrancone L. In Vivo Genetic Screens of Patient-Derived Tumors Revealed Unexpected Frailty of the Transformed Phenotype. Cancer Discovery. 2016 May 13. [Epub ahead of print] PubMed PMID: 27179036.
- Hamblett KJ, Jacob AP, Gurgel JL, Tometsko ME, Rock BM, Patel SK, Milburn RR, Siu S, Ragan SP, Rock DA, Borths CJ, O’Neill JW, Chang WS, Weidner MF, Bio MM, Quon KC, Fanslow WC. SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm. Cancer Res. 2015 Dec 2. [Epub ahead of print] PubMed PMID: 26631267.
- Herkert B, Kauffmann A, Mollé S, Schnell C, Ferrat T, Voshol H, Juengert J, Erasimus H, Marszalek G, Kazic-Legueux M, Billy E, Ruddy DA, Stump MD, Guthy D, Ristov M, Calkins K, Maira SM, Sellers WR, Hofmann F, Hall M, Brachmann SM. Maximizing the efficacy of MAPK-targeted treatment in PTENLOF/BRAFMUT melanoma through PI3K and IGF1R inhibition. Cancer Res. 2015 Nov 17. pii: canres.3358.2014. [Epub ahead of print] PubMed PMID: 26577700.
- Lantermann AB, Chen D, McCutcheon KJ, Hoffman GR, Frias E, Ruddy DA, Rakiec DP, Korn JM, McAllister G, Stegmeier F, Meyer MJ, Sharma SV. Inhibition of casein kinase 1 alpha prevents acquired drug resistance to erlotinib in EGFR-mutant non-small cell lung cancer. Cancer Res. 2015 Oct 21. pii: canres.1113.2015. [Epub ahead of print] PubMed PMID: 26490646.
- Hoffman GR, Rahal R, Buxton F, Xiang K, McAllister G, Frias E, Bagdasarian L, Huber J, Lindeman A, Chen D, Romero R, Ramadan N, Phadke T, Haas K, Jaskelioff M, Wilson BG, Meyer MJ, Saenz-Vash V, Zhai H, Myer VE, Porter JA, Keen N, McLaughlin ME, Mickanin C, Roberts CW, Stegmeier F, Jagani Z. (2014) Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers. PNAS. PMID: 24520176
- Li H, Zhang Y, Ströse A, Tedesco D, Gurova K, Selivanova G. (2014) Integrated high-throughput analysis identifies Sp1 as a crucial determinant of p53-mediated apoptosis. Cell Death Differ. PMID: 24971482