A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic β cells to secrete protective immunomodulatory factor interleukin-10. TOC GRAPHIC

replaceable with engineered cysteines, to which molecules of any size could be efficiently appended without the loss of Cas9 activity. We successfully appended biotin (small) and PEG groups (large) using thiol-maleimide chemistry. Because many possible conjugates (e.g., ssODNs) are prohibitively expensive for or unamenable to this direct thiol-maleimide conjugation, we also sought to develop a more general conjugation platform. Thus, we designed a short oligonucleotide handle 'adaptor', which is attached to Cas9 via thiol-maleimide chemistry and uses base pairing to anchor any molecule containing or appended with nucleic acids (Figure 1a). As an example of platform's utility, we used it to hybridize long ssODNs that are large, expensive, and not available in sufficient quantities for thiol-maleimide conjugation to Cas9. 15 The resulting Cas9:ssODN conjugates robustly enhanced the precise incorporation of the desired sequence from ssODN in multiple cell types and genomic sites. Importantly, the chemical conjugation platform enabled the multivalent display of ssODNs, which further enhanced the precise incorporation of the desired sequence over that of the univalent display.
Next, we demonstrated the utility of our conjugation platform by efficiently engineering insulin-producing β cells to secrete non-endogenous molecules, including an immunomodulatory protein (~160 residues), without incorporation of any viral or foreign sequences (e.g., promoter) other than that of the secreted molecule. Current β-cell transplantation therapies for type 1 diabetes suffer from immune rejection, resulting in acute cell loss and only short-term therapeutic effects. 16,17 The macroencapsulation of β cells with a semipermeable membrane can protect them from the host's immune system, though foreign body reaction-induced fibrosis can impair the mass transfer and viability of encapsulated cells. [18][19][20] Anti-inflammatory cytokines, such as interleukin 10 (IL-10), can reduce fibrosis and promotes long-term β-cell survival and superior islet function. 21-25 Therefore, engineered βcells that secrete anti-inflammatory cytokines and anti-fibrotic factors will propel the development of cell-based therapeutics for diabetes. By hijacking the insulin expression and secretion machinery, we used our herein developed conjugation platform to efficiently engineer β cells to secrete a non-endogenous peptide in a glucoseresponsive manner as is observed for insulin. Furthermore, we were able to engineer β cells to secrete IL-10, demonstrating the immediate usefulness of our conjugation platform for the development of cell-based therapeutics.

RESULTS
Multiple domains on Cas9 are tolerant of displaying molecules of diverse size and nature. To choose the sites for conjugation to Cas9, we analyzed the structures of apo-Cas9, gRNA-bound Cas9, and gRNA-and DNAbound Cas9 for residues that could provide a high labeling yield, tolerate chemical modifications, span all the domains of Cas9, and are surface-exposed in various Cas9 conformations, for efficient display of modifications ( Figure 1b). [26][27][28] Using aforementioned criteria, we identified two sites (204, 532) on the REC domain, one site (826) on the HNH domain, five sites (1,945,1026,1054, 1068) on the RuvC domain, and two sites (1153,1207) on the PI domain. We selected residues 558 and 1116 as controls since modifications at 558 will impede the Cas9:gRNA interaction and at 1116 will impede protospacer adjacent motif (PAM) recognition by Cas9 (Figures   1b and S1). We optimized the conjugation conditions for Cas9 variants using biotin-maleimide and PEG (5 kDa)maleimide as model compounds to ensure that modifications of various sizes or nature were tolerated ( Figure   S2). The reactions were fast and high yielding at all sites except for the 1153C mutant; sites proximal to 1153C (i.e., 1154C) also yielded low conjugation efficiencies ( Figure S2). The location of these residues was not assigned at the crystal structure of apo-Cas9, but the residues were assumed to be amenable to efficient conjugation, since they were expected to be surface-exposed and flexible. 26-28 Our labeling results, however, indicate that the loop may have higher-order structures that prevent efficient chemical reactions, so we did not use those sites in future experiments. To improve the compatibility of the system with a broader range of conjugates, we next utilized the optimized reaction conditions to label Cas9 at the remaining sites with a 17nucleotide (nt) DNA adaptor (5'-GCTTCACTCTCATCGTC-3'). The conversion rates were comparable to those of PEG labeling ( Figure S2), demonstrating that efficient conjugation of multiple cargo types can occur at these sites. Thus, the identified sites provide high conjugation yields with diverse molecules, including small molecule and polymers (DNA or PEG).
To identify sites that are tolerant to the conjugation of the DNA adaptor without the loss of Cas9 activity, we designed an ssODN that would insert a 33-nt DNA fragment (HiBiT sequence 29 ) at the target gene ( Figure S3).
This insertion would result in the expression of a fusion protein with a C-terminal HiBiT tag, which is a small fragment of the NanoLuc luciferase. When HiBiT is complemented by LgBiT, the remainder of NanoLuc, the fulllength luciferase is reconstituted to generate a luminescence signal proportional to the degree of knock-in, providing an easy readout for HDR ( Figure S3a). We chose GAPDH as the first target gene ( Figure S3b) owing to its abundant expression in many cell types, which should allow for the reliable detection of the luminescence signal. Using the HiBiT knock-in assay, we measured whether appending the DNA adaptor to the cysteine affected Cas9 activity ( Figure 2a). As expected, much of Cas9 activity was lost by control modifications at residues 558 and 1116, indicating the reliability of the HiBiT knock-in assay. We identified five sites in total from REC domain (532), RuvC domain (1,945,1026), and PI domain (1207) whose activity was largely maintained (>85% of wildtype in U2OS), even after labeling with the 17-nt adaptor. Finally, to investigate the off-target profile of the Cas9-adaptor conjugates, we used an eGFP disruption assay with matched gRNA and mismatched gRNAs targeting the eGFP gene, in U2OS.eGFP.PEST cells. 30,31 The Cas9-adaptor conjugate retained the target specificity while maintaining the on-target activity ( Figure S4).
Unitary display of ssODN on multiple Cas9 domains enhances HDR in several cell types. Next, we designed ssODN with a sequence complementary to the conjugation adaptor and confirmed the binding of the ssODN bearing the complementary sequence to the Cas9-adaptor using a gel-shift assay ( Figure S5). To measure the ability of ssODN conjugates to enhance HDR and site-dependence of such enhancements, we performed the HiBiT knock-in assay in U2OS cells. Using the luminescence signals from unconjugated ssODN as normalization controls, we observed enhanced knock-in efficiency at multiple sites (Figures 2b and S6a) when Cas9 displayed ssODN. We were able to confirm such enhancements in multiple cell lines, with a greater than fourfold increase in HEK-293FT cells (Figures 2c, d and S6b, c). For cells with higher HiBIT signal but lower HDR enhancements, we observed site dependence, with two internal conjugation sites (532, 945) generally performing better than the terminal conjugation site (1). An examination of the crystal structure 28 indicates that cargoes on the two internal residues are expected to align with substrate DNA, while cargoes on the terminal residue project outward from the DNA, which may explain the differences in the HDR-enhancing capacities of different ssODN-bearing sites.
The ssODN display platform allows rapid and facile screening of multiple conditions. To demonstrate the modular nature of our conjugation platform that should allow rapid testing of multiple conditions and to confirm the generality of HDR enhancement by ssODN display, we tested the ability of the conjugates to enhance HDR under several scenarios (e.g., different genomic sites, ssODN sequences, or readouts). Using the HiBiT knockin assay, we confirmed HDR enhancements at another DNA cleavage site on the GAPDH locus (Figures 3a and S7a) and at multiple genomic loci (PPIB, CFL1; Figures 3a and S7b, c). We then demonstrated HDR enhancement using a fluorescent readout and a longer knock-in fragment (GFP11, 57 nt). The correct incorporation of this fragment generated detectable fluorescence through the expression of a fusion protein with a C-terminal GFP11 tag, which forms a fully functional GFP when complemented by GFP1-10 ( Figure S8). 32 Here as well, ssODN display on Cas9 increased the knock-in efficiency by more than threefold (Figures 3b and   S7d). In addition to luminescence and fluorescence readouts to demonstrate HDR enhancement, we used a previously reported droplet digital PCR (ddPCR) assay that employs probes to distinguish between wildtype, NHEJ-edited, and HDR-edited sequences at the RBM20 locus ( Figure S9). 33 All Cas9:ssODN conjugates increased the ratio of HDR over NHEJ, again indicating the generality of our platform ( Figure 3c). The conjugates also enhanced HDR when another gRNA/ssODN pair was employed to introduce the same mutation ( Figure   S10).

Multivalent display of ssODN on Cas9 further enhances HDR over that of the univalent display.
Owing to the small size of our adaptor and the chemical nature of our platform, multivalent displays are feasible ( Figure   4a). To demonstrate the multivalent display, we produced Cas9 double-cysteine mutants (532C/945C and 532C/1207C) and labeled them with the adaptor ( Figure S11a). Next, we confirmed the binding of the ssODNs to Cas9 (Figure S11b Cas9:ssODN conjugates allow efficient engineering of β cells to secrete IL-10. To demonstrate the functional applicability of our chemically modified Cas9, we used it to efficiently engineer β cells to endow the cells with immunomodulatory function. Since C-peptide is cleaved during proinsulin processing and is co-secreted with insulin, we hypothesized that knocking in the desired gene into the C-peptide portion of the proinsulin locus would enable the secretion of the inserted gene product. Previously, a lentiviral vector encoding a proinsulin-luciferase fusion construct, containing a luciferase inserted into the C-peptide, expressed functional luciferase in levels directly proportion to insulin when stably integrated into the INS-1E β-cell line and responded sensitively to external stimuli, such as glucose concentration. 34 However, viral-vector engineering poses safety issues such as immunogenicity to viral components or the unintended random insertion of DNA fragments into the host genome. 35,36 Thus, direct knock-in of the desired gene fragment into the C-peptide using Cas9 will not require regulatory elements (e.g., promoters) and allow co-secretion of the target gene product with insulin without raising immunogenicity issues (Figures 5a and b).
To identify the appropriate insertion site in the Ins1 locus, we used HDR-mediated knock-in of the HiBiT sequence at the C-peptide portion in INS-1E cells ( Figure 5a). The target HiBiT sequence was flanked by additional prohormone convertase 2 (PC2) cleavage sites 34 to ensure that no extra amino acids would be present at each end of the knock-in product after processing ( Figure 5a). We chose three gene insertion sites at the start, middle, and terminal regions of the C-peptide locus, and designed several gRNAs to target these sites such that insertion sites and DNA cleavage sites would be close enough to obtain high HDR efficiency ( Figure 5a). In addition, genome-wide off-target profiles of gRNAs were considered such that potential off-target sites had mismatches at the seed sequences or at least three mismatches in the gene-encoding regions. When standard genome editing was performed at the target sites using non-conjugated Cas9 and ssODN, the HiBiT peptide was secreted from INS-1E cells, which could readily be detected through luminescence signals from the cell culture supernatant after complementation by the LgBiT protein. The highest knock-in efficiency was achieved by targeting the middle region of the C-peptide (site 2) (Figure 5c), so this insertion site was used for future experiments. HiBiT peptide secretion was also stimulated by glucose, indicating that the knock-in product was secreted through the insulin processing and secretion pathways (Figures 5d and S13).
Based on this optimized design, we next knocked-in Il10, whose 797-nt ssODN is much larger than that of HiBIT (183-nt). The secretory signal peptide sequence present in the Il10 gene was omitted as our approach leverages the insulin secretion pathway. PC2 cleavage sites were added at each end of Il10 to obtain intact IL-10 as the knock-in product, and the corresponding ssODN was synthesized by reverse transcription. When INS-1E cells were transfected with both unconjugated Cas9 and ssODN, IL-10 was secreted into the cell culture media as determined via enzyme-linked immunosorbent assay (ELISA). No IL-10 was detected after transfection with Cas9 or ssODN alone, or in lipopolysaccharides (LPS)-treated cells 37 (Figure 5e). We confirmed the correct insertion of the Il10 gene at the Ins1 C-peptide region using Sanger sequencing ( Figure S14). Finally, we displayed ssODN on Cas9 and found that both HiBiT secretion and IL-10 secretion were significantly promoted by Cas9-ssODN conjugation over that of separate Cas9 and ssODN (Figures 5f, g and S15).

DISCUSSION
We describe a simple, scalable, and modular chemical platform for site-specific Cas9 labeling with a wide range of functional molecules to expand Cas9 functionality for novel applications. We first identified multiple internal residues on Cas9 that can be modified using thiol-maleimide chemistry without compromising Cas9 activity in cells, opening up a variety of new site-specific conjugation locations with simple and easy-to-use chemistries.
We showed that internal conjugation sites and multivalent conjugations often had improved knock-in efficiencies, indicating the need for conjugation systems that are not limited to single modifications of the Cas9 termini. The identified sites could also be used to display inhibitors of DNA repair pathways for their local inhibition at the site of the Cas9-induced double-strand break. For example, co-administration of Cas9 with small-molecule inhibitors of the NHEJ pathway can enhance precision editing, 11 but concerns about mutagenesis stemming from genomewide NHEJ inhibition has limited the utility of such inhibitors. 38,39 Local NHEJ-pathway inhibition at the strand break site through the multivalent display of NHEJ inhibitors on Cas9 itself may allay such concerns. Such local inhibition has been demonstrated for base editors that display peptidic inhibitors of uracil DNA glycosylase for local enzyme inhibition, which improves base-editing efficiencies. 40 Similarly, the local inhibition of p53 pathway activation can increase the efficiency of precision genome editing in primary cells and stem cells where Cas9induced double-strand breaks lead to apoptosis via this pathway. 41,42 Finally, displaying tissue-specific ligands on Cas9 will enable cell-specific genome editing. 12 We also developed a short oligonucleotide handle as a universal anchoring point for any oligonucleotidecontaining functional molecules, making this platform amenable to most desired conjugates. When ssODN was attached to this anchor, HDR efficiency was enhanced in multiple cells and genomic loci, simultaneously demonstrating the utility of the conjugation technique as well as the usefulness of an increased local concentration of ssODNs for precision genome editing. Our Cas9-adaptor design is modular in that the Cas9bearing the universal adaptor can be used for any ssODN or any type of knock-in (e.g., single nucleotide exchange, short DNA insertion, and long gene insertion).
While these studies were underway, reports of genetic fusions of Cas9 to avidin, SNAP tag, or porcine circovirus 2 protein (PCV), which can also bind to donor DNAs, appeared in the literature, [43][44][45][46] and our studies complement these approaches in multiple ways. First, at 17-nt, our Cas9-adaptor constructs are much smaller than the reported constructs, with the possibility of reducing this to as low as 13 nt. Second, while these other genetic fusions are mostly tested at the N-and C-termini of Cas9, we systematically investigated both terminal and internal conjugation sites and found that internal sites yielded higher knock-in efficiencies in certain cell lines.
The ability of our system to equally address internal or terminal sites makes it significantly more flexible and adaptable to specific applications. Third, our adaptor-based conjugation strategy does not require chemical modification of the ssODNs, as opposed to avidin-or SNAP-based methods, which can be particularly costly to secret a diverse set of functional molecules, such as small peptides and large gene products. Specifically, we successfully produced cells that co-secrete IL-10, a well-established anti-inflammatory factor that reduces fibrosis and can protect β cells from pro-inflammatory cytokine-induced cell death. [22][23][24][25] This method should also enable the continuous local production of immunomodulatory factors in β cells for preventing β-cell failure, yet due to its local nature, have minimal systematic effects on the host immune system. 48 Since insulin is secreted in large quantities by β cells, only a small fraction of cells may require editing to have a therapeutic effect. Our approach of hijacking the insulin expression and secretion machinery significantly reduces the size of the exogenous sequences that needs to be knocked-in, allowing efficient engineering of the β cells using Cas9.
Moreover, our precise knock-in strategy would be safer than conventional random gene integration methods using viral vectors that are immunogenic and result in unpredictable genomic sequence. 35,36 Using this strategy, β cells could also be engineered to secrete glucagon-like peptide 1 (GLP-1), which when co-secreted with insulin may enhance the function and viability of β cells. 49 Overall, this study provides a simple and effective method for forming chemical conjugates of Cas9 to enhance its functionality, and its ease-of-use will make it convenient for scientists of all backgrounds to modify existing    Unlabeled wildtype Cas9 (wt) and Cas9-adaptors labeled at the indicated residues were used. All data from biological replicates are shown. P-values were calculated by paired two-tailed t-test.   Site-directed mutagenesis. Two cysteine residues in SpCas9 (C80, C574) were replaced by serine to give a cysteine-free mutant. Based on this construct, multiple single-cysteine and double-cysteine mutants were generated by introducing cysteines at the designated residues. Mutagenesis was performed using the partial overlapping primer design method or using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs).
Cas9 labeling by thiol-maleimide conjugation. Adaptor oligonucleotide (GCT TCA CTC TCA TCG TC) modified with protected maleimide (maleimide-2,5-dimethylfuran cycloadduct) at the 5' terminus was synthesized by Gene Link. Prior to thiol-maleimide conjugation, the maleimide group was deprotected via retro-Diels-Alder reaction by heating the DNA in toluene for 3 h at 90°C. Solvent was removed under the reduced pressure, and the resulting DNA in solid form was dissolved in water to give 2 mM solution. Cas9 cysteine mutants (4 μM) were mixed with 300 μM of PEG (5 kDa)-maleimide or adaptor oligonucleotide-maleimide in reaction buffer (20 mM TrisꞏHCl, 0.1 M KCl, 1 mM TCEP, pH 7.5). The reaction proceeded for 3 h at room temperature (RT) with mild rotation. The resulting mixture was diluted with a high-salt buffer (20 mM TrisꞏHCl, 1 M KCl, 1 mM TCEP, 20% glycerol, pH 7.5) and incubated with Ni-NTA agarose beads at 4°C. The beads were extensively washed with the high-salt buffer to completely remove non-specifically bound oligonucleotide molecules. Labeled Cas9 was eluted with an elution buffer (20 mM TrisꞏHCl, 0.1 M KCl, 1 mM TCEP, 250 mM imidazole, 10% glycerol, pH 7.5). Finally, buffer exchange was conducted using Amicon Ultra-0.5 mL centrifugal filters with a 100 kDa cut-off (Millipore) to give Cas9-adaptor conjugates in storage buffer (20 mM TrisꞏHCl, 0.1 M KCl, 1 mM TCEP, 10% glycerol, pH 7.5).

Cas9 biotin labeling and pull-down by streptavidin beads.
Cas9 with enhanced specificity [eSpCas9(1.1)] 51 was used for biotin labeling. Cas9 cysteine mutants (7 μM) were mixed with 500 μM of EZ-Link™ Maleimide-PEG 2 -Biotin (Thermo) in a reaction buffer (20 mM TrisꞏHCl, 0.1 M KCl, 1 mM TCEP, pH 7.5). The reaction proceeded for 3 h at room temperature (RT) with mild rotation. Excess compounds were removed by Bio-Gel P-6 columns (Biorad) according to the manufacturer's protocol. Next, 30 pmol of Cas9 from the above step was incubated with 30 μL of Pierce Streptavidin Magnetic Beads (Thermo) overnight at 4°C. Flow-through was collected and the beads were washed twice with a washing buffer (20 mM TrisꞏHCl, 0.15 M NaCl, 0.1% Tween20, pH 7.4; 300 μL) and once with the reaction buffer (300 μL). The beads were heated to 95°C for 5 min in the presence of SDS-PAGE buffer, and the resulting bead-bound fraction (eluate) and flow-through were subjected to SDS-PAGE followed by Coomassie staining.
Electrophoretic mobility shift assay. For this assay, 300 nM of Cas9 was mixed with 300 nM of ssODNs in a binding buffer (20 mM TrisꞏHCl, 0.1 M KCl, 1 mM TCEP, 10% glycerol, pH 7.5). For the Cas9 double-adaptor conjugates, 200 nM of protein and 400 nM of ssODN were used. For testing long ssODNs ( Figure S15e), 80 nM Cas9 and 60 nM ssODN were used. The mixture was incubated for 30 min at RT and resolved by 1% agarose gel. DNA was stained using SYBR Gold, and fluorescence images were obtained using an Azure c600 (Azure Biosystems) with the Cy3 channel.
In vitro transcription to synthesize single-guide RNAs. Sequences of target-specific forward primers and universal reverse primers are listed in Table S2. Polymerase chain reactions (PCRs) were conducted using Q5 High-Fidelity 2x Master Mix (New England Biolabs) in the presence of 0.5 µM of forward and reverse primers in a volume of 25 µL. The PCR program was as follows: Initial denaturation at 95°C for 1 min; 25 cycles of 95°C   Figure S1. Selection of Cas9 labeling sites based on crystal structures. (a) Structure of apo-Cas9 (PDB ID: 4CMP). 1 Four residues (1,532,1116,1153) are not assigned at the structure possibly due to the high flexibility. We assumed that those sites are surface-exposed based on the nucleic-acid-bound structures and/or high flexibility of the loops they belong to. (b) Structure of gRNA-bound Cas9 (PDB ID: 4ZT0). 2,3 Only residue 558 is projected toward the interior of the protein, indicating that labeling at this site can inhibit the formation of the correct ribonucleoprotein (RNP) structure. Cas9 exhibits a large conformational change, especially at the recognition (REC) lobe, upon gRNA binding (residues 204, 532, 558). PI domain is in blue, HNH domain is in yellow, RuvC domain is in green, REC domain is in red, and BH is in gray.   (c) Results of the eGFP knock-out assay. Cells were nucleofected with 10 pmol of RNP and were incubated for 48 h followed by nuclei staining and fluorescence imaging. Unlabeled wildtype Cas9 (wt) and Cas9-adaptor labeled at the indicated residues were used. Error bars represent standard deviation from ≥ four technical replicates. Figure S5. Electrophoretic mobility shift assay to check the binding between Cas9-adaptor conjugates and ssODN. When the ssODN contained the adaptor-binding sequence, the specific Cas9:ssODN complex was observed. In contrast, only non-specific binding patterns were observed when the ssODN did not have the corresponding sequence or when the unlabeled wildtype Cas9 (wt) was used. The ssODN for HiBiT knock-in at the GAPDH locus was used. Even though the lanes are not contiguous, they are all from a single gel.    The reference probe can bind to all alleles while the HDR probe binds only to the precisely edited allele. The NHEJ probe is a drop-off probe that cannot bind to the NHEJ-repaired allele. Each probe is labeled with a fluorophore-quencher pair. During the PCR, DNA-bound probes are hydrolyzed by the exonuclease activity of the DNA polymerase. Therefore, fluorophores and quenchers move apart from each other, providing fluorescence signals. Figure S10. Single base exchange at the RBM20 locus was promoted by Cas9-ssODN conjugation in HEK-293FT cells.
Unlabeled wildtype Cas9 (wt) and Cas9-adaptor labeled at the indicated residues were used. Another gRNA/ssODN pair (gRNA 2 from Table S2 and ssODN 2 from Table S3) was used. All data from biological replicates are shown. Figure S11. (a) Site-specific labeling of Cas9 mutants at two cysteine residues using thiol-maleimide conjugation. The degree of labeling was measured through SDS-PAGE followed by Coomassie staining. (b) An electrophoretic mobility shift assay was performed using Cas9-adaptors and the ssODN specific for GAPDH HiBiT tagging that contained the adaptorbinding sequence. Unlabeled wildtype Cas9 (wt) and Cas9-adaptors labeled at the indicated residues were used. The RNP and ssODN were used at a molar ratio of 1:2.    knock-in were promoted by Cas9-ssODN conjugation when two different gRNAs were tested. Unlabeled wildtype Cas9 (wt) and Cas9-adaptor labeled at residue 945 were used. All data from biological replicates are shown. (e) Electrophoretic mobility shift assay to check the binding between Cas9-adaptor and long ssODNs for Il0 knock-in. The specific Cas9:ssODN complex was observed only when both Cas9 and ssODN contained the complementary adaptor sequences. All lanes are from a single gel. Unlabeled wildtype Cas9 (wt) and Cas9-adaptor labeled at residue 945 from different batches were used.