A Type I-F Anti-CRISPR Protein Inhibits the CRISPR-Cas Surveillance Complex by ADP-Ribosylation

•Crystal structure of AcrIF11 suggests that it resembles an ADP-ribosyltransferase•AcrIF11 specifically ADP-ribosylates a key residue in the PAM-recognition loop•AcrIF11-mediated ADP-ribosylation of the Csy complex prevents dsDNA binding•AcrIF11 requires the Cas7.6f subunit for binding to and modifying the Csy complex CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems. CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems. CRISPR-Cas systems are part of the prokaryotic adaptive immune system against foreign genetic elements, including invading viruses (Marraffini, 2015Marraffini L.A. CRISPR-Cas immunity in prokaryotes.Nature. 2015; 526: 55-61Crossref PubMed Scopus (388) Google Scholar; van der Oost et al., 2014van der Oost J. Westra E.R. Jackson R.N. Wiedenheft B. Unravelling the structural and mechanistic basis of CRISPR-Cas systems.Nat. Rev. Microbiol. 2014; 12: 479-492Crossref PubMed Scopus (388) Google Scholar). They are divided into class 1 systems, which deploy multi-subunit surveillance complexes guided by CRISPR RNA (crRNA), and class 2 systems, which depend on crRNA-guided single-subunit Cas proteins. The two classes are further divided into 6 types (types I–VI) and 19 subtypes (Makarova et al., 2015Makarova K.S. Wolf Y.I. Alkhnbashi O.S. Costa F. Shah S.A. Saunders S.J. Barrangou R. Brouns S.J. Charpentier E. Haft D.H. et al.An updated evolutionary classification of CRISPR-Cas systems.Nat. Rev. Microbiol. 2015; 13: 722-736Crossref PubMed Scopus (1154) Google Scholar). The type I multi-subunit CRISPR-Cas system is the most ubiquitous and largest in molecular size, and it is further divided into seven subtypes: I-A through I-F and I-U. All type I systems deploy multi-subunit crRNA-guided surveillance complexes to identify foreign DNA, which is degraded by the trans-acting nuclease-helicase Cas3 (Cas2/3 in type I-F). The type I-F system of Pseudomonas aeruginosa encodes a 350-kDa crRNA-guided surveillance complex (i.e., the Csy complex) composed of nine Cas proteins (one Cas5f, one Cas8f, one Cas6f, and six Cas7f proteins) and a single 60-nt crRNA (Chowdhury et al., 2017Chowdhury S. Carter J. Rollins M.F. Golden S.M. Jackson R.N. Hoffmann C. Nosaka L. Bondy-Denomy J. Maxwell K.L. Davidson A.R. et al.Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex.Cell. 2017; 169: 47-57.e11Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar; Guo et al., 2017Guo T.W. Bartesaghi A. Yang H. Falconieri V. Rao P. Merk A. Eng E.T. Raczkowski A.M. Fox T. Earl L.A. et al.Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.Cell. 2017; 171: 414-426Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar; Peng et al., 2017Peng R. Xu Y. Zhu T. Li N. Qi J. Chai Y. Wu M. Zhang X. Shi Y. Wang P. et al.Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures.Cell Res. 2017; 27: 853-864Crossref PubMed Scopus (33) Google Scholar; Rollins et al., 2019Rollins M.F. Chowdhury S. Carter J. Golden S.M. Miettinen H.M. Santiago-Frangos A. Faith D. Lawrence C.M. Lander G.C. Wiedenheft B. Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.Mol. Cell. 2019; 74: 132-142Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The complex is assembled into an asymmetric spiral, in which the crRNA is largely extended, allowing direct interaction with all nine protein subunits. In the head region, Cas6f (formerly Csy4) is stably associated with the 3′ end of the crRNA after cleaving it at the stem-loop structures consisting of palindromic repeat sequences (Haurwitz et al., 2010Haurwitz R.E. Jinek M. Wiedenheft B. Zhou K. Doudna J.A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease.Science. 2010; 329: 1355-1358Crossref PubMed Scopus (449) Google Scholar; Przybilski et al., 2011Przybilski R. Richter C. Gristwood T. Clulow J.S. Vercoe R.B. Fineran P.C. Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum.RNA Biol. 2011; 8: 517-528Crossref PubMed Scopus (82) Google Scholar). Six Cas7f (formerly Csy3) subunits assemble along the crRNA, forming the backbone of the complex. In the tail region, the 5′ handle of the crRNA is anchored by extensive interactions with a stable heterodimer composed of Cas5f (formerly Csy2) and Cas8f (formerly Csy1) (Chowdhury et al., 2017Chowdhury S. Carter J. Rollins M.F. Golden S.M. Jackson R.N. Hoffmann C. Nosaka L. Bondy-Denomy J. Maxwell K.L. 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Discovery of widespread type I and type V CRISPR-Cas inhibitors.Science. 2018; 362: 240-242Crossref PubMed Scopus (86) Google Scholar; Watters et al., 2018Watters K.E. Fellmann C. Bai H.B. Ren S.M. Doudna J.A. Systematic discovery of natural CRISPR-Cas12a inhibitors.Science. 2018; 362: 236-239Crossref PubMed Scopus (75) Google Scholar), and recently type VI (Lin et al., 2020Lin P. Qin S. Pu Q. Wang Z. Wu Q. Gao P. Schettler J. Guo K. Li R. Li G. et al.CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.Mol. Cell. 2020; 78: 850-861.e5Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) CRISPR-Cas systems. For the type I CRISPR systems, 14 Acrs were described to target the type I-F system (i.e., AcrIF1-IF14), 7 Acrs were found to exclusively target the type I-E system (i.e., AcrIE1-IE7), 1 Acr targeted type I-C (i.e., AcrIC1), and 1 Acr targeted type I-D (i.e., AcrID1) (Hwang and Maxwell, 2019Hwang S. Maxwell K.L. Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.CRISPR J. 2019; 2: 23-30Crossref PubMed Google Scholar). AcrIF6 and fusion protein AcrIE4-F7 could target both type I-E and type I-F systems (Marino et al., 2018Marino N.D. Zhang J.Y. Borges A.L. Sousa A.A. Leon L.M. Rauch B.J. Walton R.T. Berry J.D. Joung J.K. Kleinstiver B.P. Bondy-Denomy J. Discovery of widespread type I and type V CRISPR-Cas inhibitors.Science. 2018; 362: 240-242Crossref PubMed Scopus (86) Google Scholar; Pawluk et al., 2016bPawluk A. Staals R.H. Taylor C. Watson B.N. Saha S. Fineran P.C. Maxwell K.L. Davidson A.R. Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species.Nat. Microbiol. 2016; 1: 16085Crossref PubMed Scopus (162) Google Scholar). Despite the growing number of identified Acr proteins, the working mechanisms of only a few of them have been characterized in detail. The structural basis underlying the mechanisms of type I-F Acrs has been determined for AcrIF1, AcrIF2 (Chowdhury et al., 2017Chowdhury S. Carter J. Rollins M.F. Golden S.M. Jackson R.N. Hoffmann C. Nosaka L. Bondy-Denomy J. Maxwell K.L. Davidson A.R. et al.Structure Reveals Mechanisms of Viral Suppressors that Intercept a CRISPR RNA-Guided Surveillance Complex.Cell. 2017; 169: 47-57.e11Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar; Guo et al., 2017Guo T.W. Bartesaghi A. Yang H. Falconieri V. Rao P. Merk A. Eng E.T. Raczkowski A.M. Fox T. Earl L.A. et al.Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.Cell. 2017; 171: 414-426Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar; Peng et al., 2017Peng R. Xu Y. Zhu T. Li N. Qi J. Chai Y. Wu M. Zhang X. Shi Y. Wang P. et al.Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures.Cell Res. 2017; 27: 853-864Crossref PubMed Scopus (33) Google Scholar), AcrIF3 (Rollins et al., 2019Rollins M.F. Chowdhury S. Carter J. Golden S.M. Miettinen H.M. Santiago-Frangos A. Faith D. Lawrence C.M. Lander G.C. Wiedenheft B. Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.Mol. Cell. 2019; 74: 132-142Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar; Wang et al., 2016aWang J. Ma J. Cheng Z. Meng X. You L. Wang M. Zhang X. Wang Y. A CRISPR evolutionary arms race: structural insights into viral anti-CRISPR/Cas responses.Cell Res. 2016; 26: 1165-1168Crossref PubMed Scopus (35) Google Scholar, Wang et al., 2016bWang X. Yao D. Xu J.G. Li A.R. Xu J. Fu P. Zhou Y. Zhu Y. Structural basis of Cas3 inhibition by the bacteriophage protein AcrF3.Nat. Struct. Mol. Biol. 2016; 23: 868-870Crossref PubMed Scopus (64) Google Scholar), and AcrIF10 (Guo et al., 2017Guo T.W. Bartesaghi A. Yang H. Falconieri V. Rao P. Merk A. Eng E.T. Raczkowski A.M. Fox T. Earl L.A. et al.Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.Cell. 2017; 171: 414-426Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). During the preparation of our manuscript, the structures of AcrIF6/8/9 complexed with the Csy complex were also reported (Zhang et al., 2020Zhang K. Wang S. Li S. Zhu Y. Pintilie G.D. Mou T.C. Schmid M.F. Huang Z. Chiu W. Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM.Proc. Natl. Acad. Sci. USA. 2020; 117: 7176-7182Crossref PubMed Scopus (12) Google Scholar). All of these Acrs inhibit the type I-F system through direct interactions with either the Csy complex (AcrIF1/2/4/6/8/9/10) or the Cas2/3 helicase-nuclease (AcrIF3). They inactivate the CRISPR system either by disrupting DNA binding (AcrIF1/2/6/8/9/10) or by inhibiting target sequence cleavage (AcrIF3). Given the diverse sequences of Acr proteins, it is important to investigate whether novel strategies are used by other Acr proteins. Here, we elucidate the mechanism of how AcrIF11 inactivates the type I-F CRISPR system. The biochemical and structural data revealed that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) that modifies N250 of the Cas8f subunit within the Csy complex from P. aeruginosa, a residue that is required for the recognition of the protospacer-adjacent motif (PAM). It has been reported that type I CRISPR-Cas systems could be used for long-range genome manipulations and expression modulations of targeted endogenous genes in human cells (Dolan et al., 2019Dolan A.E. Hou Z. Xiao Y. Gramelspacher M.J. Heo J. Howden S.E. Freddolino P.L. Ke A. Zhang Y. Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas.Mol. Cell. 2019; 74: 936-950.e5Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar; Pickar-Oliver et al., 2019Pickar-Oliver A. Black J.B. Lewis M.M. Mutchnick K.J. Klann T.S. Gilcrest K.A. Sitton M.J. Nelson C.E. Barrera A. Bartelt L.C. et al.Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells.Nat. Biotechnol. 2019; 37: 1493-1501Crossref PubMed Scopus (24) Google Scholar). Therefore, our results not only reveal an unprecedented mechanism for inactivating the type I multi-subunit CRISPR complexes but also provide an approach for designing efficient regulatory tools in the future applications of type I CRISPR-Cas systems. First, we investigated the type I-F system-targeted Acrs whose inhibition mechanisms remain elusive. Because the structural basis of inhibition has already been revealed for AcrIF1/2/3/10 and AcrIF4 has already been shown to bind the Csy complex (Bondy-Denomy et al., 2015Bondy-Denomy J. Garcia B. Strum S. Du M. Rollins M.F. Hidalgo-Reyes Y. Wiedenheft B. Maxwell K.L. Davidson A.R. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.Nature. 2015; 526: 136-139Crossref PubMed Scopus (194) Google Scholar), we purified the remaining nine Acrs, mixed them with the purified 350-kDa Csy complex from P. aeruginosa, and performed a size-exclusion chromatography (SEC) assay. We intended to identify Acrs that co-elute with neither the Csy complex nor Cas2/3 and therefore might execute their inhibition with novel strategies. All tested Acrs, except AcrIF5, AcrIF11, and AcrIF12, co-eluted with the Csy complex, indicating a strong, direct, and stable protein interaction (Figures S1A–S1I). The lack of binding of the three remaining Acrs to the Csy complex led us to test whether they inhibit the CRISPR system by interacting with Cas2/3, as AcrIF3 does (Bondy-Denomy et al., 2015Bondy-Denomy J. Garcia B. Strum S. Du M. Rollins M.F. Hidalgo-Reyes Y. Wiedenheft B. Maxwell K.L. Davidson A.R. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.Nature. 2015; 526: 136-139Crossref PubMed Scopus (194) Google Scholar). However, they did not co-elute with the purified Cas2/3 (Figures S1J–S1M). Based on the lack of evidence for direct, stable protein interactions, we speculated that the three Acrs might have evolved strategies different from the known ones (AcrIF1/2/3/4/6/8/9/10) to inhibit the type I-F CRISPR system. This manuscript focuses on the inhibition mechanism of AcrIF11, whereas the detailed inhibition mechanism of AcrIF5 and AcrIF12 will be investigated in future studies. Because of the low sequence similarity of AcrIF11 with known domains, we determined its structure (full length, 132 residues) at 3.07 Å (Figure 1A; Table 1). The structure of AcrIF11 can be divided into two lobes, a β lobe composed of mainly a six-stranded β sheet (cyan) and an α-helical lobe spanning the residues from 51 to 107 (green, Figures 1A, S2A, and S2B). During the additive screen of the optimization steps, we found that the crystals of AcrIF11 required β-nicotinamide adenine dinucleotide (β-NAD+, shortened to NAD hereafter) as an additive to show better diffraction. The final refined structure of AcrIF11 also showed a clear density for a NAD molecule located within the cleft formed on the surface of the β lobe (Figure 1A). Structural alignment using the DALI (distance-matrix alignment) server (Holm, 2019Holm L. Benchmarking fold detection by DaliLite v.5.Bioinformatics. 2019; 35: 5326-5327Crossref PubMed Scopus (168) Google Scholar) did not reveal entries with Z scores higher than 7, but the catalytic domain (C domain) of diphtheria toxin (DT) (PDB: 1TOX) was found to be the closest structural homolog of AcrIF11, with a root-mean-square deviation (RMSD) of 3.1 Å among 92 residues. Although sequence alignment indicated that the two proteins display limited homology (Figure S2C), a superimposition of the three-dimensional structures of the C domain of DT and AcrIF11 revealed considerable structural similarities (Figure 1B). DT, an ~58-kDa protein secreted by Corynebacterium diphtheriae, causes the disease diphtheria in humans by inhibiting protein synthesis (Bell and Eisenberg, 1996Bell C.E. Eisenberg D. Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide.Biochemistry. 1996; 35: 1137-1149Crossref PubMed Scopus (184) Google Scholar). The C domain of DT possesses ADP-ribosylation activity, which uses NAD as a substrate to catalyze the covalent transfer of the ADP-ribose moiety from NAD to the diphthamide (post-translationally modified His715) of elongation factor-2 (EF-2) (Bell and Eisenberg, 1996Bell C.E. Eisenberg D. Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide.Biochemistry. 1996; 35: 1137-1149Crossref PubMed Scopus (184) Google Scholar). We therefore tested whether AcrIF11 would ADP-ribosylate proteins by performing an ADP-ribosylation assay using the Csy complex or Cas2/3 as a substrate and NAD or biotin-labeled NAD (biotin is attached to the amino group of the adenine base) as co-substrates. Immunoblotting using a polyclonal pan-ADP-ribose-recognizing antibody (anti-mono-ADP-ribose and poly-ADP-ribose, specifically detecting ADP-ribosylated proteins, for experiments with NAD as a substrate) and streptavidin blots (for experiments with biotin-NAD as a substrate) both clearly revealed that the Csy complex, but not Cas2/3, is ADP-ribosylated by AcrIF11 in the presence of NAD or biotin-NAD (Figures 1C–1E). The molecular weight of the modified band approximately corresponds to the Cas8f subunit (Figures 1C–1E). Altogether, these data revealed that AcrIF11 is an ADP-ribosyltransferase that modifies the Csy complex, probably at its Cas8f subunit.Table 1Data Collection and Refinement StatisticsData CollectionAcrIF11 (SeMet)Space groupP6222Cell Dimensionsa, b, c (Å)104.677, 104.677, 119.673α, β, γ (°)90.00, 90.00, 120Resolution (Å)50–3.07 (3.21–3.07)bValues in parentheses are for the highest-resolution shell.Rsym or Rmerge (%)14.5 (113.8)I/σ(I)29.7 (2.8)Completeness (%)99.5 (96.4)Redundancy13.8 (13.0)RefinementResolution (Å)45.33–3.07No. reflections7640Rwork/RfreeaFor each structure, one crystal was used.0.2046/0.2227No. atoms1090 Protein1046 Ligand/ion44 Water0B factors79.43 Protein79.23 Ligand/ion84.22 WaterRMSDsBond length (Å)0.003Bond angle (°)0.65Ramachandran Plot (%)Favored100Allowed0Outliers0a For each structure, one crystal was used.b Values in parentheses are for the highest-resolution shell. Open table in a new tab AcrIF11 exhibits other distinct features apart from its low structural similarity with known ADP-ribosyltransferases. In the active site, the DT-like toxin family possesses a conserved HYE motif with a His residue, two Tyr residues, and a Glu residue (Figure S2D). The Glu residue, known as the key catalytic residue during the ADP-ribose transfer, is invariant even among all known bacterial ADP-ribosyltransferases (Simon et al., 2014Simon N.C. Aktories K. Barbieri J.T. Novel bacterial ADP-ribosylating toxins: structure and function.Nat. Rev. Microbiol. 2014; 12: 599-611Crossref PubMed Scopus (118) Google Scholar). However, in AcrIF11, only the His residue is conserved (H7 in AcrIF11), but the two Tyr residues and the key Glu residue are replaced by F26, H37, and D115, respectively (Figures S2D–S2F). Next, we analyzed the binding mode of NAD within the AcrIF11 structure. In the AcrIF11 structure, NAD forms a ring-like conformation in the cavity within the β lobe (Figure 2A). The nicotinamide group is stabilized by two hydrogen bonds from the carbonyl oxygen and amide nitrogen of G8 and π-π interaction with the side chain of H37 (Figure 2A). Moreover, the adenine group is stabilized by the polar interaction from the carbonyl oxygen of R17, and the ribose moiety within the ADP part is hydrogen bonded to the side chains of H7, S9, and E11. In addition, the NAD molecule is bound by hydrophobic interactions by the side chains of F26 and H117 (Figure 2A). Notably, NAD was indispensable for the inhibition of the type I-F CRISPR DNA strand cleavage in vitro by AcrIF11 (Figure 2B, lanes 4 and 5). Moreover, mutations of the NAD binding residues of AcrIF11 abolished both its ADP-ribosylation activity (Figure 2C) and its capacity of inhibiting DNA cleavage in vitro (Figure 2B). The decreased binding to NAD by the AcrIF11 mutants was confirmed using the isothermal titration calorimetry (ITC) assay (Figures 2D and S3A–S3I). A single mutation of D115 did not completely inhibit NAD binding but was enough to abolish the ADP-ribosylation of the Csy complex, suggesting that this residue is essential during the catalytic process. This is in agreement with the core catalytic Glu residue, which shares the similar spatial position in known bacterial ADP-ribosyltransferases (Figure S2F). Deletion of the α-helical lobe also caused loss of modification activity of AcrIF11, suggesting that the α-helical lobe is integral to the structure of AcrIF11 (Figure S4A). AcrIF11 was not able to deplete the NAD pool in the host cell (Figures S4B and S4C). Collectively, these data suggested that the ADP-ribosylation activity of AcrIF11 is essential for inhibiting the type I-F CRISPR system. Next, we aimed to elucidate how ADP-ribosylation would inhibit the activity of the Csy complex. First, we mapped the modification sites of the AcrIF11-modified subunit of the Csy complex. We co-expressed the Csy complex with AcrIF11, purified the Csy complex, and separated the purified complex via SDS-PAGE. Immunoblotting with the anti-ADP-ribose antibody revealed that the band corresponding to the Cas8f subunit of the Csy complex was modified by AcrIF11, whereas this was not the case when the Csy complex was expressed and analyzed alone (Figure 3A). Upon tryptic digestion, liquid chromatography-mass spectrometry (LC-MS) detected a peptide sequence, F243GGTKPQNISQLNSER258, of Cas8f with a mass addition of 541 Da, consistent with the attachment of the ADP-ribose moie