CRISPR-FOCUS: A web server for designing focused CRISPR screening experiments

Cao, Qingyi; Ma, Jian; Chen, Chen Hao; Chen, Zhi; Li, Wei; Liu, X. Shirley

doi:10.1371/journal.pone.0184281

Cited by 18 publications

(9 citation statements)

References 40 publications

(56 reference statements)

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“…A comprehensive list of sgRNA sequences projected to efficiently direct Cas9 cleavage at their target sites was generated using the sequence model using CIRSPR-FOCUS 52 and ordered as a pool of equal molar oligos ( Supplementary Table 2 ). The lentiCRISPR RBP plasmid library was cloned using previously reported methods 1 .…”

Section: Methodsmentioning

confidence: 99%

Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors

Wheeler

Einstein

et al. 2020

Nat Methods

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Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR/Cas9 screening with microRaft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; C RISPR-based microRaft, followed by gRNA Identification). By isolating microRafts that contain genetic clones harboring individual guide RNAs, we identify RNA binding proteins (RBPs) that influence the formation of stress granules, punctate protein-RNA assemblies, that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens.

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Section: Methodsmentioning

confidence: 99%

Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors

Wheeler

Einstein

et al. 2020

Nat Methods

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“…Guide RNAs were designed with CRISPR-FOCUS. 58 Oligo pool was synthesized by Synbio Technologies (Suzhou) and cloned into lentiGuide-Puro by using EasyGeno Assembly kit (VI201; TIANGEN). Pooled sgRNA viruses were used to infect CH12F3 or v-Abl cell lines stably-expressing Cas9 protein.…”

Section: Methodsmentioning

confidence: 99%

ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells

Liu

Shang

et al. 2020

Cell Res

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Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.

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“…The sgRNA target sequences were selected based on SSC score (34) and the online tool CRISPR-FOCUS (35). Each oligo (75 nt) contains 19 nt sgRNA, 5′ universal flanking sequence: TATCTTGTGGAAAGGACGAAACACCg and 3′ universal flanking sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAAT.…”

Section: Methodsmentioning

confidence: 99%

PRMT1 loss sensitizes cells to PRMT5 inhibition

Gao

Zhang

Villarreal

et al. 2019

Nucleic Acids Research

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PRMT5 is an arginine methyltransferase that accounts for the vast majority of the symmetric methylation in cells. PRMT5 exerts its function when complexed with MEP50/WDR77. This activity is often elevated in cancer cells and correlates with poor prognosis, making PRMT5 a therapeutic target. To investigate the PRMT5 signaling pathway and to identify genes whose loss-of-function sensitizes cancer cells to PRMT5 inhibition, we performed a CRISPR/Cas9 genetic screen in the presence of a PRMT5 inhibitor. We identified known components of the PRMT5 writer/reader pathway including PRMT5 itself, MEP50/WDR77, PPP4C, SMNDC1 and SRSF3. Interestingly, loss of PRMT1, the major asymmetric arginine methyltransferase, also sensitizes cells to PRMT5 inhibition. We investigated the interplay between PRMT5 and PRMT1, and found that combinatorial inhibitor treatment of small cell lung cancer and pancreatic cancer cell models have a synergistic effect. Furthermore, MTAP -deleted cells, which harbor an attenuated PRMT5–MEP50 signaling pathway, are generally more sensitive to PRMT1 inhibition. Together, these findings demonstrate that there is a degree of redundancy between the PRMT5 and PRMT1 pathways, even though these two enzymes deposit different types of arginine methylation marks. Targeting this redundancy provides a vulnerability for tumors carrying a co-deletion of MTAP and the adjacent CDKN2A tumor suppressor gene.

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CRISPR-FOCUS: A web server for designing focused CRISPR screening experiments

Cited by 18 publications

References 40 publications

Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors

Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors

ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells

PRMT1 loss sensitizes cells to PRMT5 inhibition

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