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Designing and Building Effective Pooled Libraries for Loss-of-Function Screens

(Including shRNA and sgRNA)

Pooled lentiviral-based libraries containing heterogeneous mixtures of shRNA or CRISPR sgRNA (gRNA) constructs allow you to assay the effects of many thousands of pooled constructs in one experiment. Although Cellecta’s loss-of-function screening libraries are generated using standard, proven genetic library construction techniques, there are a number of technical challenges to produce quality shRNA or sgRNA expression libraries. Cellecta’s loss-of-function constructs include both sgRNA for knockout studies using CRISPR, and shRNA for RNAi knockdown studies.

Quality Oligos

For large-scale production of heterogeneous populations of designed oligonucleotides for complex libraries, Cellecta uses array-synthesized oligos. These full-length oligonucleotides are over 100 bases in length with minimal mutations. Also, of critical importance, the solid support synthesis minimizes bias by providing similar levels of each individual species.

Well-Designed Vectors

To provide efficient delivery of complex shRNA or sgRNA libraries into different cell types for a variety of experimental designs, we have developed HIV-based lentiviral shRNA cloning vectors with U6 tet-regulated or constitutive promoters for expression of shRNA or sgRNA and a choice of a single or dual selection marker (GFP, RFP, PuroR, NeoR, HygroR, BlastR, BleoR, etc.) expressed from a single CMV, EF1, PGK, UbiC, or other promoter. Cellecta’s HIV-based lentivectors have a broad range of tropisms for efficient transduction in a wide variety of cells.

Quality pooled array-synthesized oligonucleotides and shRNA and CRISPR sgRNA (gRNA) library construction

Unambiguous Sequenceable Barcodes

Quality control of shRNA libraries and the final screening representation analysis is greatly facilitated by the incorporation of easily sequenced barcodes in each shRNA construct. The barcodes enable unambiguous identification of each shRNA species with Next-Gen Sequencing (NGS). map of Cellecta's U6 and U6-Tet lentiviral vectors

Effective shRNA

In pooled lentiviral library construction, one key challenge is the design and production of quality oligonucleotides to provide the shRNA or sgRNA inserts for the libraries. For its shRNA libraries, Cellecta has developed its own in-house shRNA design algorithm that makes use of internal studies primarily focused mostly on structural features (e.g., length, loop size, mismatches, etc.), combined with published information regarding sequence preferences, including known sequences shown effective for a particular target. shRNA sequence mismatches

Representation Levels of Individual shRNA or sgRNA Sequences

Cellecta specifically designs and constructs pooled shRNA and sgRNA (gRNA) libraries using proven library construction procedures, not by re-amplifying and mixing pre-made individual constructs. As a result, it is possible to obtain a very narrow representation of virtually all shRNA or sgRNA. The use of our “intelligent” barcoding (in the case of shRNA) in combination with NGS enables Cellecta to ensure that more than 99% of shRNA or sgRNA are present in every library. We typically achieve 80-90% representation within a 10-fold range.

Cellecta pooled shRNA library representation Upper panel shows a library with very good representation. Virtually all the shRNA are present in more than 100 copies and most are around 1,000 copies. Also, there is less than 10-fold difference between the most represented and least for about 80-90% of the shRNA, so the library has a relatively balanced representation of all shRNA. The lower panel shows a poor library where almost half of the shRNA are present at less than 100 copies and the distribution is very broad. It is only possible to get readable signals for less half the targets using the the library in the lower panel.

This definitive representation data at the start of a screen provides a starting point for the analysis to find shRNA that significantly increase or decrease during screening, indicating relevant targets. Without this starting point, it is difficult to differentiate signal vs. noise in any screening assay–or even which shRNA is actually missing in the screen. In other words, you require this data in order to determine what is truly being screened.

Quantifiable Next-Gen Sequencing (NGS)

NGS significantly outperforms the hybridization-based approach for identification of individual shRNA or sgRNA species based on the high-quality “digital expression data” generated by using barcodes (for shRNA libraries) or gRNA sequences themselves (in sgRNA libraries). Even when using optimized barcode sequences, array hybridization suffers from a limited dynamic range of approximately 2 orders of magnitude which results in a loss of as many as 30% of the signals that fall outside their effective range. Also, spot-to-spot cross hybridization on arrays results in significant noise that does not occur with NGS where virtually every shRNA or sgRNA in the population is detected and counted, from those present in only a few copies to those present in several million. Differences in shRNA or sgRNA species between control and test populations are very easily detected and statistically analyzed, so that hits can be confidently identified.

hybridization versus Next-Gen Sequencing for shRNA library screeningComprehensive Data-Rich Results

Cellecta’s pooled libraries offer the capability of targeted screens knocking out the same transcripts or genes with multiple shRNAs or gRNAs that serve to verify a specific functional effect and rule out off-target effects. In addition to unbiased wide-range screens of many thousands of genes to identify ones involved in a particular response, screens with small, highly-redundant targeted libraries provide invaluable approaches to thoroughly investigate the function of key pathways, analyze sets of known genes involved in specific responses, or validate targets identified in earlier large-scale screens.

shRNA and CRISPR Library Construction Products and Services

For information on our pooled library products and services, please visit: