Custom CRISPR and RNAi Pooled Libraries
By taking advantage of array-based oligonucleotide synthesis, we can readily make precisely defined large custom pooled libraries expressing many thousands of elements. Within approximately 3 months, we can produce a completely new, high quality custom library. The library vector can also be easily customized, and the same library oligonucleotides can be cloned into more than one vector backbone (e.g. with an inducible and constitutive shRNA or sgRNA promoter). It is a very flexible platform.
Cellecta has the expertise and capability to generate high quality complex heterogeneous libraries consisting of virtually any defined sequences. This capability combined with our proprietary vectors and lentiviral expertise provides Cellecta with the ideal platform to create excellent quality, highly representative pooled genome-wide and targeted lentiviral sgRNA CRISPR Custom libraries, shRNA Custom libraries for RNAi screens and Libraries for Barcoding Cells.
Cellecta Libraries feature
- A higher percentage of functional shRNAs, designed using validated shRNA sequences and optimized shRNA structures
- Flexible array-based oligonucleotide synthesis enables rapid creation of complex libraries expressing any set of shRNAs and sgRNAs to any targets
- Optimized shRNA and sgRNA expression vectors with various markers, selections, and both inducible and constitutive promoters
- Lentiviral-based system ensures efficient delivery of high complexity, pooled custom shRNA and CRISPR libraries into a wide range of cell types
- Compatible with Illumina HiSeq, GAIIx, and NextSeq NGS platforms
For more information on how Cellecta builds our pooled shRNA and sgRNA expression libraries, please see our Technology section.
How Many shRNAs or sgRNAs Do You Recommend Per Transcript? How Effective is the Knockdown/ Knockout?
How Many shRNAs or sgRNAs Do You Recommend Per Transcript? How Effective is the Knockdown/ Knockout?
For broad coverage, we usually design libraries so there are at least 5-6 shRNAs or sgRNAs targeting a specific transcript. Our approach to shRNA design draws on in-house design and structural studies. We can design libraries to target each transcript with as many shRNAs or sgRNAs as desired. Some labs are interested in designing shRNA or sgRNA to “tile” target transcripts. We have optimized the shRNA structure (e.g., length, loop size, mismatches, etc.) and sgRNA structure so that they produce highly effective target knockdown or knockout and are amplified and maintained effectively in a pooled library. On average, more than 65% of the shRNAs knock down transcript levels by greater than 70%. More than 90% of cells expressing Cas9 and sgRNA knock out transcript levels after about 9 days of expression.
What Sizes of Custom shRNA and CRISPR Libraries Do You Offer?
We provide custom-designed shRNA or sgRNA libraries targeting any set of genes of interest. Since we utilize array-based synthesis for library inserts, the oligonucleotide pool sizes are somewhat fixed.
- Typical library sizes range anywhere from 1,000 to 80,000 shRNAs
- Targeting up to genome-wide coverage
- For specialized applications such as in vivo screening, we can provide small libraries (targeting <1,000 transcripts or genes)
Custom Library Service
The library construction service includes oligo design, oligo synthesis, library construction, and QC by clone sequencing and NGS. Deliverables include 500 μg plasmid DNA library, the plasmid cloning vector, library sequences, clone sequencing QC data, and representational analysis with NGS data.
- shRNA library prices start from $25,000
- CRISPR sgRNA library prices start from $21,000
Cellecta can package the libraries and provide them in ready-to-transduce pseudoviral format at additional cost. Packaging of the Cas9 construct for CRISPR libraries can also be provided.
Vector Design for Custom shRNA and CRISPR Libraries
Our standard library vector expresses shRNA or sgRNA from a constitutive U6 promoter and contains puromycin antibiotic selection and a red fluorescent protein marker (as shown). However, as indicated in the figure, we can provide customized changes to any of these elements, including using an inducible promoter. We find that, for library screening in cell culture, inducibility adds an additional variable that can complicate the assay unnecessarily (more information about this is available in our blog entry, “RNAi Screening with An Inducible Promoter: Is There an Advantage?”). However, for specialized applications, we can certainly provide libraries with these inducible H1 or U6 promoters upon request.
For more information on loss-of-function screening, visit the Library Technology page.
If you are interested in off-the-shelf, whole human genome CRISPR or shRNA or mouse shRHNA pooled lentiviral libraries, please visit our Products page.
Next-Gen Sequencing of Samples from Genetic Screens
Cellecta shRNA and 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 barcodes or sgRNA sequences
- Performs NGS on the Illumina NextSeq or HiSeq
- Enumerates shRNA or 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
Please email email@example.com to obtain a quotation.
|Catalog #||Item Description||Price|
|SVC9-PS||pR-CMV-Cas9-2A-Hygro Cas9 Expression Vector (plasmid), 25 μg||$300|
|SVC9-VS||pR-CMV-Cas9-2A-Hygro Cas9 Expression Vector (virus), 1 x 106 TU||$500|
|SVC9B-PS||pR-CMV-Cas9-2A-Blast Cas9 Expression Vector (plasmid), 25 μg||$300|
|SVC9B-VS||pR-CMV-Cas9-2A-Blast Cas9 Expression Vector (virus), 1 x 106 TU||$500|
|CPCP-K2A||Ready-to-Use Lentiviral Packaging Plasmid Mix, 250 μg|
(for (25) 10-cm plates)
|LFVC1||LentiFuge™ Viral Concentration Reagent, 1 ml|
(for 1 liter supernatant)
|CRTEST||CRISPRtest™ Functional Cas9 Activity Kit (Human)|
(for 5 cell line pairs)