<figure><img src="image_1.png" /></figure>

HAL
open science

# mSphere of Influence: Expanding the CRISPR Sphere with Single-Locus Proteomics

Lucy Glover

<figure><img src="image_2.png" /></figure>

## To cite this version:

Lucy Glover. mSphere of Influence: Expanding the CRISPR Sphere with Single-Locus Proteomics. MSphere, 2020, 5 (1), 10.1128/mSphere.00001-20. pasteur-03107138

HAL Id: pasteur-03107138

https://pasteur.hal.science/pasteur-03107138

Submitted on 12 Jan 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_3.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_4.png" /></figure>

# mSphere of Influence: Expanding the CRISPR Sphere with Single-Locus Proteomics

Lucy Gloverª

ªDepartment of Parasites and Insect Vectors, Institut Pasteur, Paris, France

**ABSTRACT** Lucy Glover's research focuses on the role of DNA repair and recombination in antigenic variation in the parasite *Trypanosoma brucei*, the causative agent of both human and animal African trypanosomiasis. In this mSphere of Influence article, she reflects on how "A CRISPR-based approach for proteomic analysis of a single genomic locus" by Z. J. Waldrip, S. D. Byrum, A. J. Storey, J. Gao, et al. (Epigenetics 9:1207-1211, 2014, https://doi.org/10.4161/epi.29919) made an impact on her research by taking the precision of CRISPR-Cas9 and repurposing it to look at single-locus proteomics. By using this technology in trypanosomes, Dr. Glover and her colleagues could study the dynamic accumulation of repair proteins after specific damage and gain insight into how the location of a double-strand break (DSB) dictates repair pathway choice and how this may influence immune evasion in these parasites.

**KEYWORDS** *Trypanosoma brucei*, antigenic variation, VSG, DNA repair and recombination, CRISPR-Cas, DNA repair, recombination

It appears that if you can imagine it, CRISPR can do it. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome modification is a type II CRISPR system from *Streptococcus pyogenes* that is the basis for prokaryote adaptive immunity (1) and that has quickly become a precision genome editing tool targeting either DNA or RNA (2) with a myriad of potential uses. Cas9 in its active form is an endonuclease that cleaves DNA and is able to bind to its target via a guide RNA (gRNA). However, by introducing mutations into the two Cas9 nuclease domains, researchers were able to retain the binding capacity of Cas9 but without DNA cleavage (3). This resulted in a CRISPR-deactivated Cas9 (dCas9) system that can be used to alter the transcription status of specific loci (4) or live-cell imaging of specific loci (5) and to interrogate long-range chromatin interactions (6). In the article "A CRISPR-based approach for proteomic analysis of a single genomic locus" by Waldrip et al. (7), the authors used CRISPR-dCas9 to develop CRISPR-chromatin affinity purification with mass spectrometry (ChAP-MS) technology to determine the changes in the local epiproteome, defined as posttranslational modifications (PTMs) of histones and other proteins, of a promoter during activation of transcription. Using CRISPR-ChAP-MS, the authors observed enrichment of chromatin from the *GAL1* gene promoter under activating conditions and identified a specific cohort of proteins associated with transcription. Although in our lab we do not study promoter activation, we are interested in the accumulation of proteins involved in DNA damage and repair after a double-strand break (DSB) and how this influences the ability of *Trypanosoma brucei* to exchange genes, specifically the variant surface glycoprotein (VSG) genes, required for immune evasion. Of the kinetoplastid parasites, *T. brucei* is the most well studied and genetically tractable (8), making new technologies potentially applicable to studies in this parasite. Could we use CRISPR-ChAP-MS to target specific loci after a DSB to interrogate the dynamics of repair and recombination?

Chromatin immunoprecipitation (ChIP)-MS has long been established as a method to define chromatin composition at specific regions in the cell (9), but it relies on a known

**Citation** Glover L. 2020. mSphere of Influence: expanding the CRISPR sphere with single-locus proteomics. mSphere 5:e00001-20. https://doi.org/10.1128/mSphere.00001-20.

**Copyright** © 2020 Glover. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Lucy.glover@pasteur.fr.

The views expressed in this article do not necessarily reflect the views of the journal or of ASM.

**Published** 22 January 2020
<figure><img src="image_5.png" /></figure>

protein for immunoprecipitation. CRISPR-ChAP-MS extends ChAP-MS and TAL (TALEN [transcription activator-like effector nuclease])-ChAP-MS approaches, both of which use quantitative high-resolution mass spectrometry to unambiguously identify specific PMTs and proteins localized to the region of interest. This technology uses dCas9 tagged with protein A (PrA) and targeted with a gRNA specific to the GAL1 gene promoter region. The ability to activate the GAL1 gene promoter in a synchronous manner by supplying Saccha-romyces cerevisiae with galactose instead of glucose allowed the authors to target tran-scriptionally active chromatin for analysis. Formaldehyde-fixed, sheared chromatin was affinity purified using the PrA tag and subjected to label-free mass spectrometry. The CRISPR-ChAP-MS method was shown to have two benefits: (i) the improved unbiased target chromatin isolation at 1-kb resolution and (ii) the ease with which gRNAs can be designed for multiplexing, making this approach cost-effective. One disadvantage is that both CRISPR-ChAP-MS and TAL-ChAP-MS, which require binding to the DNA, are unlikely to be able to target repressed regions of chromatin.

Homologous recombination (HR) is a dynamic process that is essential for main-
taining genomic integrity and for immune evasion in trypanosomes. Antigenic variation in
trypanosomes is mainly via VSG gene conversion (GC) events, which are dependent on HR.
We know that DSBs can trigger a VSG gene switch and that several DNA damage proteins
are important for this process, but what constitutes this dynamic repair factory that forms
and leads to switching is unknown (10). There are several features that could make
implementing the method as described in the Waldrip et al. paper (7) in trypanosomes, to
look at accumulation of DNA repair proteins, particularly attractive. First, similar to activa-
tion of the GAL1 promoter, we are able to induce a single DSB in 95% of the population
using the I-Scel meganuclease (10), which allows us to spatially and temporally control the
accumulation of DNA damage repair proteins. Second, a single VSG gene is expressed from
a specialized locus, called an expression site, at the telomeric end of a chromosome, making
the chromatin we are interested in open. Third, DNA repair and recombination are dynamic
processes meaning that we could assay the composition of the repair factory over multiple
time points using the same gRNAs. The one challenge we would face is finding multiple
potential gRNA sequences for tilling without relying wholly on a drug resistance cassette
inserted into the target site, as in the trypanosome genome, there are approximately 20
expression sites which exhibit a high degree of homology. However, this is a problem that
could be overcome. Understanding how trypanosomes switch the expressed VSG gene is
not fully understood even though it is fundamental to their immune evasion strategy. A
method that could allow us to sample, in an unbiased manner, the factors that leads to VSG
gene switching is an exciting one.

ACKNOWLEDGMENT

L.G. is supported by an ANR JCJ (VSGREG; ANR-17-CE12-0012) grant and Institut Pasteur G5 position.

REFERENCES

1. Marraffini LA. 2015. CRISPR-Cas immunity in prokaryotes. Nature 526: 55-61. https://doi.org/10.1038/nature15386.

2. Koonin EV, Makarova KS, Zhang F. 2017. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol 37:67-78. https://doi.org/10.1016/j.mib.2017.05.008.

3. Dominguez AA, Lim WA, Qi LS. 2016. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol 17:5-15. https://doi.org/10.1038/nrm.2015.2.

4. Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. 2013. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 8:2180-2196. https://doi.org/10.1038/nprot.2013.132.

5. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li G-W, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B. 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479-1491. https://doi.org/10.1016/j.cell.2013.12.001.

6. Fujita T, Fujii H. 2013. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding

molecule-mediated chromatin immunoprecipitation (enChIP) using
CRISPR. Biochem Biophys Res Commun 439:132-136. https://doi.org/10
.1016/j.bbrc.2013.08.013.

7. Waldrip ZJ, Byrum SD, Storey AJ, Gao J, Byrd AK, Mackintosh SG, Wahls WP, Taverna SD, Raney KD, Tackett AJ. 2014. A CRISPR-based approach for proteomic analysis of a single genomic locus. Epigenetics 9:1207-1211. https://doi.org/10.4161/epi.29919.

8. Matthews KR. 2015. 25 years of African trypanosome research: from description to molecular dissection and new drug discovery. Mol Biochem Parasitol 200:30-40. https://doi.org/10.1016/j.molbiopara.2015.01.006.

9. Wierer M, Mann M. 2016. Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes. Hum Mol Genet 25: R106-R114. https://doi.org/10.1093/hmg/ddw208.

10. Sima N, McLaughlin EJ, Hutchinson S, Glover L. 2019. Escaping the immune system by DNA repair and recombination in African trypanosomes. Open Biol 9:190182. https://doi.org/10.1098/rsob.190182.