A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells

Dalvai, Mathieu; Loehr, Jérémy; Jacquet, Karine; Huard, Caroline; Roques, Céline; Herst, Pauline M.; Côté, Jacques; Doyon, Yannick

doi:10.1016/j.celrep.2015.09.009

Cited by 100 publications

(152 citation statements)

References 63 publications

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“…Cell Line 4D-Nucleofector X KitLonzaCat# V4XC1012 Deposited Data Raw data (ChIP-seq and BLESS)This paperArrayExpress: E-MTAB-5817RAD51 and XRCC4 ChIP-seqAymard et al., 2014ArrayExpressE-MTAB-1241RNA polymerase II S2P ChIP-seqCohen et al., 2018ArrayExpressE-MTAB-6318MethylCap-seqDeplus et al., 2014GEO GSE26810 Experimental Models: Cell Lines DIvA cellIacovoni et al., 2010N/AU20S-ISceI GFP-RFP (NHEJ)Jacquet et al., 2016N/A3xFlag-Twin-strep-tagged SUPT7L K562Dalvai et al., 2015N/AU2OSN/AATCC HTB-96, RRID:CVCL_0042 Oligonucleotides HR-DSB1 for ChIP-qPCRFW GATTGGCTATGGGTGTGGACREV CATCCTTGCAAACCAGTCCTAymard et al., 2014N/AHR-DSB2 for ChIP-qPCRFW CCGCCAGAAAGTTTCCTAGAREV CTCACCCTTGCAGCACTTGAymard et al., 2014N/ANHEJ-DSB for ChIP-qPCRFW TGCCGGTCTCCTAGAAGTTGREV GCGCTTGATTTCCCTGAGTAymard et al., 2014N/AACTB for ChIP-qPCRFW AGCCGGGCTCTTGCCAATREV AGTTAGCGCCCAAAGGACCAThis paperN/ATAF12 for ChIP-qPCRFW GCTGAGACGAACGCTTCACTREV CCTTCGAACACTGACCCACTThis paperN/AsiRNA siSUPT7L-46 CUACUAGACCCAACAGAAA[dT] [dT] UUUCUGUUGGGUCUAGUAG[dT] [dT]This paperN/AsiRNA siSUPT7L-47 CUAUCACAGUUACAUGCUA[dT] [dT]UAGCAUGUAACUGUGAUAG[dT] [dT]This paperN/AsiRNA siKAT2B (PCAF) CUCUAAUCCUCACUCAUUU[dT] [dT]AAAUGAGUGAGGAUUAGAG[dT] [dT]This paperN/AsiRNA siKAT2A (GCN5) GCUACUACGUGACCCGGAA[dT] [dT]UUCCGGGUCACGUAGUAGC[dT] [dT]This paperN/A Recombinant DNA pX330-LMNAgRNA1Pauty et a...…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

et al. 2018

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SummaryDouble-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

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Section: Methodsmentioning

confidence: 99%

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

et al. 2018

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“…Third, the smallest input we tested (10 8 cells) still exceeds the amounts obtained from experiments such as flow sorting (10 5 to 10 6 cells). Further scaling will depend on optimizing bait expression and incorporation within chromatin, potentially with Cas9-mediated introduction of the BioTAP-tag into the endogenous gene (32), and on improving peptide detection and fragmentation efficiency within the mass spectrometer.…”

Section: Discussionmentioning

confidence: 99%

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Zee

Alekseyenko

McElroy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.chromatin complexes | affinity pulldown | liquid chromatography-mass spectrometry | BioTAP-XL | histone modifications C hromatin encompasses the subset of proteins and RNAs in complex with DNA to form the chromosomes within eukaryotic nuclei. As the primary protein constituents of chromatin, histones organize genomic DNA into nucleosomes and display an extensive array of posttranslational modifications (PTMs) (1). It is increasingly evident that specific histone PTMs are selectively recognized by specific nonhistone proteins that are themselves regulators of many nuclear events such as epigenetic silencing (2, 3). Consequently, defects in these chromatin components often manifest in a number of human diseases (4).As specific interactions between modified histones and nonhistone proteins help distinguish complexes participating in distinct processes, the identification of protein and modification-dependent interactions provides key insights into chromatin biology. Various chromatography and pulldown methods have been developed to identify binding partners. In a typical native pulldown experiment, nuclei from hypotonically lysed cells are digested with micrococcal nuclease (MNase). Extracted chromatin is subjected to immunoprecipitation to isolate the desired complexes. If using a tag such as FLAG, complexes are then competitively eluted and identified. Several drawbacks to native pulldowns as described here are that MNase leaves behind a substantial insoluble nuclear pellet and degrades associating RNAs, that salt extraction of the digested nucleosomes risks dissociation of the tagged bait from its interacting partners, and that most tags do not have sufficient affinity for their respective antibody to permit highly stringent washes. The result is often an incomplete picture of the chromatin...

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“…This led us to investigate whether we could further boost its activity. First, we added an N-terminal nuclear localization signal (NLS) to a previously described human codon-optimized expression construct 35 and established a K562 cell line stably expressing St1Cas9 (S. thermophilus strain LMD-9) from the AAVS1 safe harbor locus 36,37 ( Fig. 1a and Supplementary Fig.…”

Section: Identification Of An Sgrna Architecture Directing Robust Dnamentioning

confidence: 99%

“…We deposited the most active version of this vector (v3) (pAAV_LP1B_St1Cas9_LMD-9_SpA_U6_sgRNA; Addgene plasmid # 110624). To establish clonal K562 cell lines constitutively expressing C-terminally tagged SaCas9 and St1Cas9 under the control of an hPGK1 promoter, the Cas9 ORFs from pX602 and MSP1594_2x_NLS were subcloned into AAVS1_Puro_PGK1_3xFLAG_Twin_Strep 37 (Addgene plasmid # 68375). Untagged AcrIIA proteins were expressed transiently from a modified pVAX1 vector (Thermo Fisher Scientific) containing a beta-globin intron.…”

Section: Genome Editing Vectorsmentioning

confidence: 99%

“…Cells (2E5 per transfection) were transfected using the Amaxa 4D-Nucleofector (Lonza) per manufacturer's recommendations. K562 cell lines expressing SaCas9 and St1Cas9 from the AAVS1 safe harbor locus were generated as described 36,37 . Briefly, simultaneous selection and cloning was performed for 10 days in methylcellulose-based semi-solid RPMI medium supplemented with 0.5 µg/ml puromycin starting 3 days post-transfection.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

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Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

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The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

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A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells

Cited by 100 publications

References 63 publications

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

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