5C). We thank Hua B. Bai for cloning plasmids used in the TXTL assay, Shawn M. Ren for help with preliminary cell line construction, Fuguo Jiang for a gift of AcrIIA4 protein, Gavin Knott for a gift of AcrVA4 protein, the Bondy-Denomy laboratory for sharing their lab space, and the J.A.D.
We further focus our search efforts to MGEs only, where nearly all Acrs have been discovered (2), reducing the search space to a few hundred kilobases or less.
At 24 h postinduction, transgenic GFP genome editing efficiency and inhibition thereof were quantified by flow cytometry (Attune NxT, Thermo Fisher Scientific).
Genes corresponding to bivalent states were not expressed, and bivalent loci showed lower levels of methylation than polycomb and heterochromatin silenced regions. Cell lines were not subsequently authenticated or tested for mycoplasma except for the T98G cell line, which was verified to be mycoplasma free by a PCR assay. Will the 2020s Be the Decade of Life Science.
The selection of self-targeting genomes (7, 10, 19) is meant to address this issue by narrowing screening efforts to only a few candidate genomes. “We also need to understand how CRISPR effectors are behaving in live cell environments. Although candidate 10, hereafter referred to as AcrIIA15, does not occur in a CRISPR self-targeting strain, a close homolog (80% identical via blastp) can be found in Staphylococcus pseudintermedius strain 104N, which is self-targeting (SI Appendix, Fig. It seems to be either the interdisciplinary and collaborative nature of the lab, or the desire to apply science to a meaningful set of problems, to see real-world impact. Each of these constructs was grown in Rosetta2 cells overnight in lysogeny broth and subcultured in 750 mL of terrific broth. AcrIIA15 was able to inhibit both SpyCas9 and SauCas9 in human cells, but to a much lesser extent. S2). Although several studies have quantified single histone modifications in GBM-derived cell lines, none of these studies have been performed in uncultured primary tissue, and few have looked at patterns derived from multiple histone modifications in the same cell line or tumor (3). All other data discussed in the paper are available in the main text, SI Appendix, and Datasets S1 and S2. To understand which genes are regulated by which states, we used the GENCODE (10) annotation, version 24, and used bedtools closest to identify the distance between chromatin states and the closest protein-coding gene(s).
From the above merged consensus peaks for each experiment, we used ChromHMM v1.12 (5) to build a 21-state model, using only tumor ChIP-seq data. Yet, an even more direct method is to use a guilt-by-association search approach, which leverages the repeated observations that Acrs tend to coexist in similar operons and that the genomic neighborhood of several Acrs contain anti-CRISPR-associated (aca) genes that are closely coupled with Acrs and widespread in bacteria and MGEs. S5A). For lentiviral particle production on six-well plates, 1 µg lentiviral vector, 0.5 µg psPAX2 and 0.25 µg pMD2.G were mixed in 0.4 mL Opti-MEM, followed by addition of 5.25 µg PEI. Reactions were performed by adding Acrs after complexing Cas9 and sgRNA (Top) or prior to the addition of sgRNA (Bottom), as indicated to the Left of each panel. Tumors were collected during planned surgical resections, and only excess tissue that was not used for pathologic analysis was used in this study. S5A).
AcrIIA14 might also trigger complex dimerization to form an inactive conformation, similar to the mechanisms of AcrIIC3 (31, 32) or AcrVA4 (33) (Fig. As any scientist knows, not all of their ideas are a sure bet.
This work was funded in part by grants from the Cancer Prevention Research Institute of Texas (RP120194) and NIH (HG004563, CA130075, and CA198648 to V.R. S5B).
4A; Supplementary Fig. These motifs were largely degenerate and bore resemblance to motifs for several families of transcription factors, such as KLF and STAT-like binding motifs (Supplementary Fig. 4A). Data were normalized on samples without guide RNA expression treated ±doxycycline (1 µg/mL). The raw fastq files from the GBM datasets generated and analyzed in this study are available in the dbGaP repository (http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001389.v1.p1). The resulting libraries were run on an Illumina HiSeq 2500 as above. B, A polycomb-repressed region, marked by H3K27me3, in the same tumor as A over the gene KRT72, which encodes a keratin protein. Viral supernatants were filtered using 0.45 µm polyethersulfone (PES) membrane filters, diluted in cell culture media if appropriate, and added to target cells. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Available datasets and their sequencing details are summarized in the “Overview” and “ChIP-seq” sheets of Supplementary File S1.
laboratory for thoughtful comments and feedback. Briefly, the STSS was used in the NCBI database for all assemblies returned using a search of “Staphylococcus[organism] NOT phage NOT virus.” Each assembly was checked for instances of self-targeting, which were reported along with details about the instance including: target site mutations, up/downstream target sequences, Cas genes found near the array of the self-targeting spacer, predicted CRISPR subtype based on repeats/Cas proteins, repeat sequences, and conservation, etc.
To determine the stage at which the Acrs are able to inhibit SauCas9 activity, we performed the SauCas9 in vitro cleavage experiments again, but added the Acr proteins before the addition of sgRNA. RNA was isolated from homogenized tumor tissue using TRIzol (15596-026, Thermo Fisher Scientific). CRISPR has huge potential for a lot of global challenges that we are facing to date — some of them from climate change to human diseases, loss of biodiversity. A total of 840 of these genes showed strong enrichment for pattern specification, Wnt signaling, embryonic development, transcription factor DNA-binding domains (Fig. Based on our data, the AcrIIA13, AcrIIA14, and AcrIIA15 proteins we identified are likely fusions between each of three unique Acr proteins and a shared aca gene in the form of an Acr gene that inhibits Cas9 with an aca gene fused to the N terminus that is dispensable for DNA cleavage inhibition. We then designed 27 genome fragment (GF) amplicons in total, covering all of the predicted sequences, each up to ∼10 kb, with the goal of best capturing complete operons wherever possible (SI Appendix, Table S2). 6C). Copyright © 2020 the Author(s). Jennifer Doudna is a world-renowned biochemist at the University of California, Berkeley, best known for her foundational research on CRISPR, a gene editing technology that has transformed biological research in less than a decade. S2A). To determine if AcrIIA13–AcrIIA15 are capable of binding to the promoter-proximal IR sequences, we performed DNA EMSAs, combining each purified Acr protein with a fluorescein amidite (FAM)-labeled dsDNA sequence spanning the IRs in the promoter-proximal region of AcrIIA15 (Fig. The authors would also like to thank Kimberly Bowles, Ph.D., Brad Reveal, Ph.D., and Haridha Shivram for critical reading of the manuscript and Dr. Raymond Jones for helpful advice. The cultures were then pelleted and resuspended in 500 μL of phosphate buffered saline (PBS). (D) Individually purified Acr proteins bind to the AcrIIA15 promoter-proximal sequence containing inverted repeats, causing an upshift. B, Heatmap of gene expression from the 307 enhancer-associated genes with a functional annotation in A, demonstrating clustering of proneural and MES/CL tumors with regard to gene expression.
All cleavage reactions were then quenched on completion with 2 µL of 6× quenching buffer containing 30% glycerol, 1.2% SDS, 250 mM EDTA. The frequent clustering of Acr and aca genes together allows for a “guilt-by-association” approach that quickly identifies potential Acr candidates for experimental testing, but requires a known Acr or aca gene to seed the search (5⇓–7, 9, 15). This motif is a prominent feature of aca genes, which were recently shown to negatively regulate the transcription of their own operon (16, 35).
Nearly all currently known Acrs can be found in MGE sequences, especially phages, highlighting their use as a CRISPR counterdefense mechanism (3). Homogenized tumor tissue was aliquoted by weight into 15 ml conical tubes, and suspended in PBS + 10 μg/mL PMSF in isopropyl alcohol, with 1% formaldehyde for cross-linking. B, A view of 877 kb on chromosome 1 for the tumor GBM8, showing 7 ChIP-seq tracks, RNA-seq, chromatin states, and gene annotations. 6B; Supplementary Fig. Performing ChIP-reChIP to conclusively identify active and repressive marks in the same cells could resolve this ambiguity, but the large number of cells required makes such experiments infeasible in clinical tissue samples. Candidate Acr proteins were tested in a HEK293T-based monoclonal reporter cell line called HEK-RT1 (36, 37), which features a doxycycline-inducible GFP reporter (43) that can be edited before doxycycline-based induction of its expression. HEK-RT1 cells, a human HEK293T-based genome editing reporter cell line with a doxycycline-inducible GFP, were sequentially transduced with lentiviral vectors expressing Acr candidates, SauCas9 or SpyCas9, and guide RNAs targeting GFP or a negative control. To identify potential Acrs that inhibit SauCas9, we first used the Self-Target Spacer Searcher (STSS) (19) to query all Staphylococcus species deposited in the National Center for Biotechnology Information (NCBI) database for instances of CRISPR self-targeting.
This approach was repeated using any neighboring proteins to further locate their neighbors. Thus, the tumors that we used for chromatin profiling represent authentic and clinically relevant GBM subtypes.
(B) Promoter-proximal sequences for AcrIIA13–AcrIIA15 showing the presence of two sets of IRs. It is possible that AcrIIA14 either causes the SauCas9-sgRNA RNP to form higher order structures like AcrVA4 (33) or AcrIIC3 (31, 32), or interacts with RNP to prevent cleavage but not binding like AcrIIC1 (31).
J.A.D.
9) in such a way as to preserve sample identity.
Iyer).
The resulting cell lines were then further transduced with a vector expressing a GFP-targeting or negative control sgRNA and an associated mCherry fluorescence marker.
A lot of people’s best ideas are not necessarily at their desk or at their bench, but just chatting with people at softball or talking with people at the bar.”, For obvious reasons, Jennifer Doudna’s lab is widely acknowledged as one of the best CRISPR labs in the world.
Enhancers in mesenchymal and classical tumor subtypes drove gene expression associated with cell migration and invasion, whereas enhancers in proneural tumors controlled genes associated with a less aggressive phenotype in GBM. The AcrIIA13b homolog that lacks the N-terminal DNA binding domain showed a comparable trend of SauCas9 selective inhibition. 3 A, Left and SI Appendix, Fig. These aca genes encode proteins that bind to promoter-proximal sequences containing two IRs to block polymerase access.
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