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.
CRISPR technology exploits a bacterial system that slices up DNA from invading viruses to specifically target and permanently disrupt (“knock out”) genes in mammalian cells. Targeting a gene for knockout using CRISPR simply requires a short strand of RNA (sgRNA) that contains a region matching part of the gene sequence of interest, and the Cas9 nuclease protein. As a result, sgRNA-expressing constructs can be made to target and knock out essentially any gene, and as shown in the 2014 Science publications by Wang T, et al., and Shalem et al., collections of these sgRNA constructs interrupt expression of thousands of genes throughout a cell population. The cells can then be screened to look for the appearance or elimination of various phenotypes, such as viability, pathway activation, or the appearance of cell surface markers.
Pooled lentiviral-based libraries containing heterogeneous mixtures of CRISPR sgRNAs (or gRNA) constructs allow you to assay the effects of many thousands of knockouts in one experiment. 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.
In addition, screens have been done with variations of the CRISPR system with modified Cas9 proteins that, rather than disrupt the genomic DNA and completely knock out the target gene, instead affect the level of expression of the target gene. With these modified CRISPR systems, screens can be run where gene targets are selectively inhibited (CRISPRi) or activated (CRISPRa) to assess the effect on phenotypes of interest. The libraries for CRISPRa and CRISPRi screens require similar sgRNA structures, but with a design that targets the promoter regions. In addition to ready-to-use, genome-wide CRISPRi and CRISPRa libraries, Cellecta also provides custom libraries for CRISPRi and CRISPRa screens.
Generating Custom CRISPR Libraries
You provide us with your list of targets, and Cellecta does the rest!
We design oligonucleotides encoding sgRNAs to your target genes. We incorporate 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. In addition, we also use our improved sgRNA scaffold structure which incorporates the HEAT modifications to the non-targeting tracrRNA sequence on the sgRNA molecule.
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 when analyzing screening results.
Once the library is made, we isolate a few dozen constructs for full insert sequencing to confirm the configuration of the sgRNA expression cassette and ensure correct insertion; then we also deeply sequence all guides by NGS, to confirm full representation of the oligo pool, and assess distribution. Libraries that don’t 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
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.
The whole process takes approximately two months once the gene list is finalized.
Please email email@example.com to obtain a quotation.
Pooled shRNA Libraries
- RNAi Pooled Lentiviral shRNA Libraries (Online Manual or PDF)
NOTE: This User Manual replaces all previous shRNA library manuals.
CRISPR sgRNA Libraries
- CRISPR Pooled Lentiviral sgRNA Libraries (Online Manual or PDF)
Custom Library Vectors
The following shRNA and CRISPR sgRNA cloning vectors are our standard custom library vectors. If interested in a vector not listed, please see the Vector Information page.
shRNA Library Vectors
|Constitutive Library shRNA Knockdown Vectors||NGS Cassette*||Sequence**||Map|
|pRSI16cb-U6-(sh)-13kCB18-UbiC-TagRFP-2A-Puro (for clonal barcode libraries only)||HTS6cb||.txt|.gbk|.dna||Map|
|pRSI17cb-U6-(sh)-13kCB18-UbiC-TagGFP2-2A-Puro (for clonal barcode libraries only)||HTS6cb||.txt|.gbk|.dna||Map|
|Inducible Library shRNA Knockdown Vectors||NGS Cassette*||Sequence**||Map|
|pRSIT16cb-U6Tet-(sh)-13kCB18-CMV-TetRep-2A-TagRFP-2A-Puro (for clonal barcode libraries only)||HTS6cb||.txt|.gb|.dna||Map|
CRISPR sgRNA Library Vectors
|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|
Safety Data Sheet (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