Guide to Plasmid Pooled Libraries

Barcoding

Barcode libraries contain large numbers of plasmids, each with a short, randomized, unique nucleotide sequence, or barcode, used to mark individual cells. These barcodes will then be inherited by any daughter cells, making barcode libraries ideal for lineage tracing. Barcode libraries can be used for clonal fitness and competition assays, measuring bottlenecks after treatments, deconvolution of pooled perturbations, and linking perturbations to single-cell phenotypes. These libraries are also commonly used to monitor the dynamics of heterogeneous tumors.

CRISPR

CRISPR is a useful tool for genetic screening experiments, due to the relative ease of designing gRNAs and the ability to modify virtually any genetic locus. CRISPR pooled libraries deliver many gRNAs at once, targeting many genes in one experiment. These libraries may be designed to target all the genes in the genome of an organism or a subset of genes, such as those involved in a certain pathway or biological process. In a CRISPR screening experiment, target cells are treated with the pooled library to create a population of mutant cells that are then screened for a phenotype of interest. Screening experiments using a pooled CRISPR library are far more complex than using CRISPR to modify a single genomic locus. CRISPR pooled libraries are also useful for loss-of-function screens, gain-of-function screens, enhancer/promoter mapping, and variant effect mapping studies. You can learn more about CRISPR tools in our CRISPR Guide.

There are multiple types of CRISPR libraries:

• Knockout: CRISPR knockout libraries are designed to create insertions or deletions in targeted genes across the genome, rendering them nonfunctional.
• Activation: CRISPR activation libraries direct an inactive Cas enzyme fused to a transcriptional activator to target genes or regulatory regions, where it stimulates transcription by recruiting activators or modifying local chromatin.
• Inhibition: CRISPR inhibition libraries direct an inactive Cas enzyme (alone or fused to a transcriptional repressor) to target genes, where it represses expression by blocking transcription initiation or modifying local chromatin.
• Base Edit: CRISPR base edit libraries use gRNAs to guide base editors to convert one base to another.
• Prime Edit: CRISPR prime edit libraries use prime editing guide RNAs (pegRNAs) to target genes for creating insertions, deletions, and base edits.
• Other: CRISPR libraries have also been designed for other specific purposes, such as barcoding or targeting RNA.

Screening

Screening libraries (non-CRISPR) can be used for many different high-throughput experiments. They are useful for probing cellular pathways and mechanisms in gain- or loss-of-function screens, in vivo mutagenesis, pathway mapping, and drug resistance studies.

Surface Display

Surface display libraries use a genetic engineering technique that physically links a protein's function (phenotype) to the gene that encodes it (genotype). This is achieved by fusing the gene of interest with a gene encoding for an anchoring protein. The resulting fusion protein is then expressed on the surface of a host cell or virus, such as bacteriophages or yeast.

Surface display libraries are useful for epitope mapping, protein engineering, and ligand discovery. The key advantage of this approach is the ability to screen large, diverse libraries for specific binding properties without the need to purify individual proteins. Any successful candidate can be readily identified by recovering and sequencing its associated genetic material.

Commonly used surface display platforms include:

• Phage Display: In this system, a peptide or protein is fused to a bacteriophage coat protein. The resulting phages can be screened for binding affinity to a target molecule.
• Yeast Display: In this system, proteins are displayed on the surface of Saccharomyces cerevisiae, often by fusion to the Aga2p protein. This platform supports the correct folding and post-translational modification of complex eukaryotic proteins, such as antibodies.

cDNA/ORF Overexpression

cDNA libraries are created from an organism’s actively transcribed mRNA, as opposed to genomic libraries, which are created from genomic DNA. Since cDNA libraries lack introns and other nontranscribed sequences, they are smaller and easier to use than genomic libraries, but they do not contain as much regulatory information. To make a cDNA library, cDNA is synthesized, ligated to adapters, and then cloned into a given vector. They are useful for gain-of-function screens, pathway discovery, and functional complementation.

Amplifying and Using the Library

Once you receive your library from Addgene, check the library information to see if it should be amplified before you conduct your screen. If you need to amplify the library, please refer to the depositor’s protocol for the best results. If you have any questions about the library protocols, feel free to contact us at [email protected] for assistance.

For some libraries, plasmid DNA can be delivered directly to the cells of interest. With others, notably pooled lentiviral plasmid libraries, the plasmids must first be used to make viral vectors. This pooled viral vector library is subsequently used to deliver the plasmids to the cells of interest. In either case, NGS of the amplified library DNA is recommended to verify that the library contains all of the expected plasmids — an incomplete library may lead to false positives or false negatives in later experiments, and can also negatively affect data reproducibility.

Types of Pooled Library Screens

In pooled lentiviral library screens, cells are transduced at a very low multiplicity of infection (MOI) to increase the odds that an transduced cell receives only one plasmid. To make sure that every plasmid is adequately represented in the population, you’ll need to transduce many more cells than the number of plasmids present in the library – for example, the Moffat lab uses 200x more cells than plasmids in the TKO library for their screen. Using large numbers of cells minimizes the effects of random chance that could lead to false positive or negative results – for a given plasmid, think of each cell carrying it as being a biological replicate for that plasmid.

Library screens can be divided into two types: positive screens and negative screens. Both types of screen employ a selection method relevant to the phenotype being studied. An example of a published selection mechanism for lentiviral CRISPR libraries is resistance to the nucleotide analog 6-thioguanine. Because NGS is required to accurately identify which library elements are affected by selection, access to this technology is essential for any pooled library screen.

Both positive and negative screens follow the same basic process. Before applying to cells, the library must be amplified (electroporation and maxiprep) and packaged into lentiviral vectors (if delivering by viral vectors). The screens diverge at a few important points, including when to apply selection, when to sequence, and how to analyze the results.