Custom CRISPR sgRNA Pooled Libraries
In addition to our ready-to-use, off-the-shelf Genome-Wide CRISPR libraries, Cellecta offers a custom CRISPR library construction service.
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 10 years working with pooled shRNA 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
Process to Make 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
After the design step, we synthesize and clone the pool of oligos in any of our standard vectors 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 fo the oligo pool, and assess distribution. Libraries that don’t meet our standards are remade.
On completion, we provide 500
- all sequence information on the sgRNA guides and vector
- the cloning site design
- primer information for sequencing
- NGS 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.
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
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Custom shRNA Libraries
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- 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
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