The CRISPR/Cas9 system has quickly become an important tool for the genome engineering community, but the technique has primarily been limited to focused gene knock-outs and direct transfection protocols. The vectors and sgRNA libraries described below expand upon the CRISPR family of plasmids by A) offering a lentivirus-based mechanism for sgRNA delivery and B) providing a means for large scale functional screens.

Before ordering, please review the steps for using pooled lentiviral CRISPR libraries and keep the following in mind:

• These libraries are pooled. Most of these screens that can be done with the libraries will require access to next generation sequencing technology.

• These libraries are lentiviral in nature, so please ensure that you are equipped and authorized to make and use lentivirus. Also, note that due to the size of the backbone, transformations require electroporation.

Jonathan Weissman Lab (targets human genes)

The libraries described in the following article use inactive dCas9 to activate (CRISPRa) or inhibit (CRISPRi) gene transcription in human cells. The CRISPRa sgRNA library uses the sunCas9 system and contains 10 sgRNAs for each transcription start site in 15,977 human genes, and a set of 5,968 control sgRNAs for a total of 198,810 sgRNAs. The CRISPRi library contains 10 sgRNAs for each transcription start site in 15,977 human genes, and a set of 11,219 control sgRNAs for a total of 206,421 sgRNAs. The parental vector for the CRISPRa and CRISPRi libraries is pU6-sgRNA EF1Alpha-puro-T2A-BFP.

Feng Zhang Lab (libraries targeting human and mouse genes)

SAM library

The synergistic activation mediators (SAM) library is now available.

CRISPR/Cas9 Synergistic Activation Mediator (SAM) is an engineered protein complex for the transcriptional activation of endogenous genes. The SAM library consists of 3 unique sgRNAs targeting each human RefSeq coding isoform in the proximal promoter (> 90% of sgRNAs are targeted to the first 200bp upstream of the transcription start site of their target). The total library size is 70,290 guides. For SAM gain-of-function screening, this sgRNA library has to be combined with two additional SAM constructs – dCas9-VP64 and MS2-P65-HSF1.
These are provided along with the library.

GeCKO v2 library

The human genome-scale CRISPR-Cas9 knockout (GeCKO) v2 library consists of 122,417 unique guide sequences targeting 19,052 human genes and including 1000 control (non-targeting) sgRNAs. The murine library consists of 130,209 unique guide sequences targeting 20,661 murine genes. There are two versions of each library. The lentiCRISPR v2 still expresses both Cas9 and the sgRNA on the same plasmid but can produce 10-fold higher titers than the original lentiCRISPR backbone. The 2 vector format uses lentiGuide-Puro as a backbone and produces an even higher titer but must be used with cells that already contain Cas9. The lentiCas9-Blast vector is provided with this 2-plasmid version of the GeCKO library.

David Sabatini Lab / Eric Lander Lab (targets human genes)

The library described in this article consists of 73,151 sgRNA plasmids that cover a total of 7,114 human genes and include 100 non-targeting controls. The library has been separated into 6 distinct enriched sub-pools: 4 pools enriched for gene targets of known function (kinases, cell cycle proteins, nuclear proteins, and ribosomal proteins), 1 pool that targets genes of unknown function, and 1 control pool. Note that though each sub-pool is enriched for their respective target sgRNAs, they are not exclusive and elements of the entire library may be present in each sub-pool.

There are two lentiviral vectors associated with this paper, one expressing Cas9 (pCW-Cas9) and the sgRNA backbone (pLX-sgRNA).

Kosuke Yusa Lab (targets mouse genes)

The library described in this article consists of 87,897 unique gRNA plasmids, targeting 19,150 mouse protein coding regions. The library is designed for lentiviral expression in mouse cells.

Feng Zhang Lab (targets human genes)

The genome-scale CRISPR-Cas9 knockout (GeCKO) library originally described in Shalem*, Sanjana*, et al., Science 2014. The original library is discontinued and an updated version of this library is now available.

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