Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

Morgens, David W.; Wainberg, Michael; Boyle, Evan A.; Ursu, Oana; Araya, Carlos L.; Tsui, C. Kimberly; Haney, Michael S.; Hess, Gaelen T.; Han, Ke; Jeng, Edwin E.; Li, Amy; Snyder, M; Greenleaf, William J.; Kundaje, Anshul; Bassik, Michael C.

doi:10.1038/ncomms15178

Cited by 323 publications

(363 citation statements)

References 57 publications

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“…1,32,33 Additionally, a fourth paper leverages this phenomenon to assess off-target cutting. 34 Therefore, there is now substantial evidence that cell viability is determined at least in part by the number of DNA breaks per cell. A second effect on viability could be off-target activity at an essential gene.…”

Section: Resultsmentioning

confidence: 99%

Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs

et al. 2018

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The CRISPR-Cas9 system provides unprecedented genome editing capabilities. However, off-target effects lead to sub-optimal usage and additionally are a bottleneck in the development of therapeutic uses. Herein, we introduce the first machine learning-based approach to off-target prediction, yielding a state-of-the-art model for CRISPR-Cas9 that outperforms all other guide design services. Our approach, Elevation, consists of two interdependent machine learning models—one for scoring individual guide-target pairs, and another which aggregates these guide-target scores into a single, overall summary guide score. Through systematic investigation, we demonstrate that Elevation performs substantially better than competing approaches on both tasks. Additionally, we are the first to systematically evaluate approaches on the guide summary score problem; we show that the most widely-used method performs no better than random at times, whereas Elevation consistently outperformed it, sometimes by an order of magnitude. We also introduce an evaluation method that balances errors between active and inactive guides, thereby encapsulating a range of practical use cases; Elevation is consistently superior to other methods across the entire range. Finally, because of the large scale and computational demands of off-target prediction, we have developed a cloud-based service for quick retrieval. This service provides end-to-end guide design by also incorporating our previously reported on-target model, Azimuth. (https://crispr.ml:please treat this web site as confidential until publication).

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Section: Resultsmentioning

confidence: 99%

Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs

et al. 2018

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“…To identify new regulators of both constitutive and induced cell surface PD-L1 expression, we performed a whole genome CRISPR/Cas9 deletion library screen5 in the pancreatic cancer cell line BxPC-3, which displays endogenous PD-L1 that can be further induced with IFN-γ (Figure 1a and Extended Data Fig. 1).…”

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confidence: 99%

CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity

Burr

Sparbier

Chan

et al. 2017

Nature

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Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell mediated immunosurveillance1,2. The success of therapies that disrupt PD-L1 mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression1. Using a genome-wide CRISPR/Cas9 screen we identified the uncharacterized protein CMTM6 to be a critical regulator of PD-L1 in a broad range of cancer cells. CMTM6 is a ubiquitously expressed, protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome we find that CMTM6 displays remarkable specificity for PD-L1. Importantly, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC Class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour specific T-cell activity in vitro and in vivo. These findings provide novel insights into the biology of PD-L1 regulation, identify a previously unrecognised master regulator of this critical immune checkpoint and highlight a new potential therapeutic target to overcome immune evasion by tumour cells.

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“…1b). For the screen, we transduced clonal L1-G418 R cells with a lentiviral genome-wide sgRNA library such that each cell expressed a single sgRNA 10 . We then dox-induced the cells to turn on the L1-G418 R reporter for retrotransposition, and split the cells into G418-selected conditions and unselected conditions, which served to eliminate cell growth bias in the screen analysis.…”

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confidence: 99%

Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

Liu

Lee

Swigut

et al. 2017

Nature

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263

365

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Summary Transposable elements (TEs) are now recognized not only as parasitic DNA, whose spread in the genome must be controlled by the host, but also as major players in genome evolution and regulation1,2,3,4,5,6. Long INterspersed Element-1 (LINE-1 or L1), the only currently autonomous mobile transposon in humans, occupies 17% of the genome and continues to generate inter- and intra-individual genetic variation, in some cases resulting in disease1,2,3,4,5,6,7. Nonetheless, how L1 activity is controlled and what function L1s play in host gene regulation remain incompletely understood. Here, we use CRISPR/Cas9 screening strategies in two distinct human cell lines to provide the first genome-wide survey of genes involved in L1 retrotransposition control. We identified functionally diverse genes that either promote or restrict L1 retrotransposition. These genes, often associated with human diseases, control the L1 lifecycle at transcriptional or post-transcriptional levels and in a manner that can depend on the endogenous L1 sequence, underscoring the complexity of L1 regulation. We further investigated L1 restriction by MORC2 and human silencing hub (HUSH) complex subunits MPP8 and TASOR8. HUSH/MORC2 selectively bind evolutionarily young, full-length L1s located within transcriptionally permissive euchromatic environment, and promote H3K9me3 deposition for transcriptional silencing. Interestingly, these silencing events often occur within introns of transcriptionally active genes and lead to down-regulation of host gene expression in a HUSH/MORC2-dependent manner. Together, we provide a rich resource for studies of L1 retrotransposition, elucidate a novel L1 restriction pathway, and illustrate how epigenetic silencing of TEs rewires host gene expression programs.

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Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

Cited by 323 publications

References 57 publications

Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs

Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs

CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity

Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

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