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Seven must-read CRISPR screening papers

It’s hard to keep up with the rapidly expanding world of CRISPR, and it’s starting to feel like CRISPR screens are being published every week, taking the technique from the cutting edge to the mainstream.

If you’d like to understand a bit more about CRISPR screens, here’s a number of fantastic publications that have really moved this technology forward. 

CRISPR Screens... the beginning!

Not one but two papers to begin with, the two papers that first documented the use of CRISPR-Cas9 for genetic screening: Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells – Shalem et al and Genetic Screens in Human Cells Using the CRISPR-Cas9 System – Wang et al. These were published back to back in Science in December of 2013 from the labs of luminaries in the field, Feng Zhang, David Sabatini and Eric Lander (all at the Broad Institute). Both papers use the identification of essential genes as the proof of concept, following which they demonstrate the use of the technology for resistance screening – for gene knockouts that confer resistance to 6-TG and vemurafenib respectively.

…moving beyond DNA cleavage

It wasn’t long before scientists realized that the recruitment of a protein to a specific genomic site could be used to mediate changes beyond just cuts in the DNA. By taking a catalytically inactive form of Cas9 and fusing it to other effectors of DNA genes could be transcriptionally controlled.

Gilbert et al in their October 2014 paper Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation took the concepts of CRISPR inhibition (CRISPRi) and CRISPR activation (CRISPRa) and scaled them to the genome level, creating libraries with 10 sgRNAs targeting the transcription start sites of every gene in the genome. These methods move CRISPR screening beyond just the coding regions, and promise to be transformative in assessing the full breadth of transcripts encoded by the human genome.

…changing the way we think about library design

For any CRISPR screen, design of an effective sgRNA library is key. Early libraries were designed with most guides close to the start codon of the genes they’re targeting, with the idea being that frameshift mutations would result and the gene would be rendered non-function. Unfortunately, some mutations result in in-frame variants, and some genes have alternative start codons or splice variants that can circumvent frameshifts.

In June 2015 Shi et al demonstrated in their paper - Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains – that by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains they could generate a higher proportion of null mutations, and significantly improve the potency of the negative selections – thus moving forward thinking on best practice for CRISPR knockout library design.

…in primary immune cells

In July 2015, Parnas et al published A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks. This was the first work using such technology in primary immune cells, which are not as easy to handle as cell lines. Immune cells have evolved to react to every stimulus they get so their manipulation for such big experiments is not as simple. The immunology values from this study can be huge.

…pushing the technology in vivo

Since CRISPR screening’s inception Feng Zhang’s group have demonstrated a variety of applications. In this paper - Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis – Chen et al used the approach to identify genes in cancer cell metastasis, by transducing cells with their lenti-CRISPR library, implanting these transduced cells subcutaneously, and then recovering metastases and sequencing the sgRNAs therein.

…improving screen resolution

Early library designs generally used 6 sgRNAs per gene for screening. In their December 2015 paper - High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities - Hart et al took lessons learned from the first generation CRISPR screens above to design a high-complexity, genome wide sgRNA library containing 12 guides per protein coding gene totaling 176,500 guides (plus a further 1400 control guides).

They used this library to dramatically expand (by almost five times) the catalog of human core and context-dependent fitness genes (any gene whose perturbation decreases cell growth and proliferation).

So where next?

Increasingly CRISPR screens are being used as the starting point for scientific projects – unbiased screens from which hypotheses can be drawn and subsequently tested. The quality of the data that CRISPR screens produce puts PhD students and Postdocs on an excellent footing for their 3-4 year tenures, or is pointing pharmaceutical companies at some very interesting and potentially novel drug targets.

If you’re interested in bringing CRISPR screens into your research we’d like to help! At Horizon we’ve built on our background in synthetic lethality screening using siRNA, and developed a complete CRISPR screening pipeline from library design, through NGS and bioinformatics to hit nomination.