Towards targeted Cas9 (CRISPR‐Cas) delivery: Preparation of IgG antibody‐Cas9 conjugates using a split intein

The CRISPR‐Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease‐protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9‐RNP. We opted for protein trans‐splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine‐less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG‐Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double‐strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N‐terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG‐Cas9 provides the first step towards a novel CRISPR‐Cas9 targeting approach involving the preformed RNP complex.


| INTRODUCTION
The efficient manipulation of genes has always been a central driving force of biological research. 1,2More recently, specific genome editing has been accomplished even in complex eukaryotic cells and organisms, for applications from basic research to investigate gene function up to therapeutic approaches to repair genetic defects in humans.The CRISPR-Cas9 system 3 is generally considered as the most powerful to specifically introduce the required DNA double-strand breaks (DSB).However, a key challenge for expanding the application to multicellular organisms and ultimately human patients is the cellspecific delivery of the gene editing devices. 1 The CRISPR-Cas9 system is derived from bacteria and archaea, where it is part of an adaptive immune system targeting viral and foreign DNA. 4 The essential advantage over other systems for gene editing is the simplicity of programming it to the desired genetic locus.Its two components, an engineered sgRNA (a covalent chimeric fusion of the pre-crRNA and tracrRNA), complementary to the target DNA, and the nuclease protein (Cas9, Class II), form a ribonuclease-protein (RNP) complex. 1,3o establish the genome-editing complex, DNA, mRNA, and sgRNA, or protein and sgRNA can be introduced into cells.Alternatively, the preassembled RNP complex is delivered through various strategies. 1,5Delivery of the preformed RNP complex produces fewer off-target effects than the delivery via plasmids carrying the genetic information for Cas9 and the sgRNA. 6Furthermore, its cellular decay results in a shorter half-life time and avoids the risks of permanent effects from the incorporation of DNA encoding a bacterial protein.
Ex vivo delivery methods into isolated cell cultures or primary cells are manifold and include, for example, electroporation of RNP complexes, which is impracticable for in vivo administration.In vivo viral delivery offers high transduction efficiency and can display some tissue selectivity, but comes with virus-associated safety hazards. 5,7,8r non-viral delivery of different forms of nanoparticles some spatial control spatial control can be achieved through local injections. 8,9r all these delivery vehicles a programmable targeting method to cell types of choice is urgently sought after. 10Conjugating a ligand for the asialoglycoprotein receptor (ASGPr) to the RNP complex has allowed its specific targeting to cells presenting the receptor and subsequent gene editing, demonstrating that the RNP complex can survive the uptake and shuttling through the endosomes. 11tibodies are the prime reagents for cell-specific targeting in a more general manner through various cell-specific epitopes.As a prominent example, antibody-drug conjugates (ADCs) are nowadays established therapeutics to deliver their conjugated toxic cargo following specific recognition of cell-specific surface-presented antigens. 12Internalizing antibodies as tumor targeting devices have also been prepared by protamine bioconjugation and electrostatic assembly with anionic siRNA 13,14 or an ibrutinib derivate 15 into vesicular nanocarriers.However, an IgG-Cas9 covalent conjugate, as a vehicle for cell-specific delivery of the CRIPSR-Cas system, has not been reported yet.This is likely because the expression requirements for each part in a simple genetic fusion protein are not compatible (see below).
7][18][19] They enable a wide variety of protein engineering applications when employed in a heterologous protein context.In particular, they can be used for protein conjugation by posttranslationally linking two protein parts.Split inteins have been reported for the splicing of small-molecule modifications 20 or a bacterial toxin 21 to an IgG.dCas9 has been chemically modified with conjugated small-molecule probes by protein trans-splicing. 22ile several split inteins have been selected and engineered for rapid and robust protein trans-splicing, [23][24][25][26][27] we have recently reported the first cysteine-less split inteins as preparatively useful protein ligation tools. 28,29In contrast to other split inteins, which use one or two cysteine residues for forming the two (thio)ester intermediates of the protein splicing pathway, 30,31 these inteins employ two serine residues at the respective positions (Figure 1).By obviating the need to reduce the cysteines for efficient splicing, cysteine-less split inteins can be used in the absence of reducing agents, which is beneficial, for example, to keep disulfide bonds in the protein of interest (POI) intact.Furthermore, the short N-terminal split intein precursor fragments (Int N ) of the CL and PolB16 split inteins, consisting of only 26 and 15 amino acids, respectively, facilitate their preparation by solid-phase peptide synthesis (SPPS) in order to then transfer chemical moieties incorporated in their flanking region to a protein of interest in the Int C -POI precursor. 28,29 this work, we report for the first time the preparation of an IgG-Cas9 conjugate by protein trans-splicing between the heavy chain (HC) of selected monoclonal IgG antibodies and Cas9.We show that the ligated Cas9 formed the RNP complex with sgRNA in vitro and that the resulting IgG-RNP conjugate was active in introducing a DSB in its target DNA in vitro.These results represent the first steps towards a cellular delivery protocol of the preformed RNP complex of the CRISPR-Cas9 system using antibodies as targeting moieties.

| General
Solvents and standard chemical reagents were purchased from Sigma Aldrich, Acros Organics, TCI, Alfa Aesar, Carbolution, Fluorochem, Iris, or Merck and were used without further purification.Restriction enzymes were purchased from Thermo Scientific.Synthetic DNA strings were ordered from Thermo Fisher.Synthetic oligonucleotides and sgRNAd were ordered from Biolegio.Plasmids were verified by DNA sequencing by Seqlab.

| Protein production and purification
Cas9 fusion proteins were produced in Escherichia coli BL21(DE3) Gold cells.Cells were cultured at 37 C in LB-medium with the corresponding antibiotic until an OD 600 of 0.6-0.8 was reached.Protein F I G U R E 1 Reaction scheme of protein trans-splicing to link two proteins with a peptide bond.Highlighted are side chains of the key amino acids at the splice junctions, which can be cysteine, serine or threonine.Using cysteine-less inteins (X O, R = H or CH 3 ) no reducing agents are needed for full activity and hence structures in the flanking proteins sensitive to reduction, for example, disulfide bonds, can be preserved.
expression was induced at 20 C for 20 h by adding IPTG (0.4 mM, pET-based vector systems).Cell pellets were collected by centrifugation, resuspended in the respective purification buffer, flash frozen, and stored at À20 C until further use.Cell pellets were resuspended in Ni-NTA buffer (50 mM Tris/HCl, 300 mM NaCl, pH 8.0) and were ruptured using an Emulsiflex C5 emulsifier (Avestin).Insoluble fractions were removed by centrifugation and the supernatant fractions were used to purify the proteins.Ultimately, the protein was purified via size-exclusion chromatography (SEC).For this, the protein solution was injected onto a HiLoad 16/600 Superdex 200 prep grade column at 4 C using an ÄKTA Purifier (GE Healthcare).The protein was eluted at a flow rate of 1 ml/ min.Fractions were collected and concentrated.Purified and pooled protein fractions were dialyzed three times against a HEPES potassium buffer (HPB: 20 mM HEPES, 500 mM KCl, pH 7.5), of which the first contained 1 mM DTT additionally, and finally dialyzed against HPB with 10% glycerol before flash freezing in liquid nitrogen and storage at À80 C. Protein concentrations were determined using the calculated extinction coefficient at 280 nm.

| Expression and purification of antibody-Int N fusion constructs
Open reading frames were cloned into pVitro plasmid backbone and transfected by linear poly-ethylene-imine (PEI), 1 cultivated in Power-CHO Medium (Lonza) for 10 days.The cellular supernatant was sterile filtrated and subjected to affinity chromatography of protein G sepharose (Cytiva) on Äkta automated systems and eluted by pH shift to glycine-HCl pH 2.5.The eluate was adjusted to PBS buffer by gel filtration and used for splicing assays.

| Protein trans-splicing assays
Splicing assays were performed in HPB using the described concentrations and at the specified temperatures.The splicing reaction was initiated by mixing N-and C-terminal intein precursors.The reaction was stopped at the described time points by taking an aliquot of the reaction mixture and boiling (5 min, 98 C) the aliquots in 4Â sodium dodecyl sulfate (SDS) sample buffer (500 mM Tris/HCl, 8% (w/v) SDS, 40% (v/v) glycerol, 20% (v/v) 2-mercaptoethanol, 5 mg ml À1 bromophenol blue, pH 6.8).

| Densitometric analysis and determination of rate constants
Coomassie-stained SDS gels were scanned and the signal intensity of Coomassie-stained bands was determined using ImageJ.The signal intensity was normalized to the molecular weight of the protein.The ratio x of the normalized intensities of the splice product and precursor proteins was calculated and inserted in the following equation to determine the splice yield:

| Thiol bioconjugation for protein labeling
Purified SBP-Int C -Cys-H 6 precursor (10 μM) in PBS was diluted in splice buffer to a concentration of 25 μM and incubated with EDTA

| Nuclease assays
The protocol for this assay was taken and adjusted from Anders and Jinek. 2 In a first step, the nuclease, Cas9, and sgRNA (BRAF-V600E: UAG CUA CAG AGA AAU CUC GA) were incubated at equimolar concentration (1 μM) in nuclease buffer at 37 C for 10 min to form the ribonucleoprotein (RNP) complex.As a negative probe, the same Cas9 probe was incubated without the sgRNA.After the incubation the same volume of diluted target DNA (pRGS-BRAF, 4 ng/μl) in nuclease buffer was added to yield a final concentration of 0.5 μM RNP and 2 g/μl target DNA. 2 The assay mixture was incubated for 1 h at  and Int C split intein precursor proteins, both parts would be ligated in the protein trans-splicing reaction.Another advantage of this approach is that the split intein precursor fragments are removed during the protein trans-splicing reaction, thus enabling a virtually traceless conjugation.However, we expected also protein trans-splicing to be very challenging given enormous size of the protein components resulting in a combined size of about 470 kDa.Thus, we aimed to address the question whether a split intein could be utilized for this task.

| Genetic fusion and expression of IgG and Cas9 split intein precursor proteins
We selected the recently described cysteine-less split CL intein 28 to splice the antibody and Cas9 parts.This cysteine-independent intein does not require a treatment with reducing agents like DTT or TCEP to increase splicing yields, neither during isolation and purification of the split intein precursor fusion proteins nor during the actual protein trans-splicing reaction.Hence, an undesired interference with the disulfide bonds of the antibody, that may lead to at least partial reduction and subsequent disulfide scrambling, 12 can be avoided.Genetic fusions were prepared to append the Int N fragment (26 aa) of the intein C-terminally to the HC of the antibody, and the Int C fragment (129 aa) N-terminally to Cas9.For each intein fragment, five native flanking amino acids were included to increase the chances of high splicing efficiency.
We chose three antibodies of clinical relevance, the expression of which was previously well established in our laboratories, namely, the two anti-IGF1R antibodies cixutumumab and teprotumumab for treatment in human Ewing sarcoma 13,32 and an anti-CD33 antibody for treatment of human acute myeloid leukemia. 14The antibodies with the respective HC-Int N fusion constructs were produced in CHO cells and purified by affinity chromatography using protein G sepharose to give antibody precursor constructs cixutumumab-Int N (construct 1; see Figure 2), teprotumumab-Int N (construct 2; see Figure S4) and anti-CD33-Int N (construct 3).
The Cas9 gene of Streptococcus pyogenes 33 was designed as synthetic DNA with optimized codon usage for E. coli and expressed as a fusion gene to give H 6 -SUMO-Int C -Cas9-SBP (construct 4).A flexible linker sequence at the N-terminal end of Cas9 was included to provide sufficient distance from the IgG HC in the final spliced conjugate.
SBP was used for a second affinity purification on streptactin beads following the initial Ni-NTA chromatography to give highly pure protein (Figure S1).

| Semi-synthetic splice assays to prove split intein precursor activity
To assay the splicing activity of the antibody and Cas9 split intein precursors, we first tested their individual splicing with excess amounts of fluorescently labeled intein precursor counterparts.Int C -Cys (construct 5; full sequence SBP-Int C -CysTag-H 6 ) contains a single cysteine in its short C-extein sequence (CysTag) that was bioconjugated with 5 (6)-carboxyfluoresceine (Cf) using maleimide chemistry to give SBP-Int C -CysTag (Cf)-H 6 (construct 5*). 28,34,35On the other hand, Cf-Int N (construct 6) was prepared by solid-phase peptide chemistry as a synthetic peptide (31 aa) with Cf coupled to the N-terminal end of the 5 flanking N-extein residues.
The HC of the cixutumumab antibody was converted to $84% (Figure 2B), while the H 6 -SUMO-Int C -Cas9-SBP precursor (4) was nearly completely consumed (Figure 2D).Both splice products cixutumumab-CysTag (Cf)-H 6 and Cf-Cas9-SBP could also be clearly identified on SDS-polyacrylamide gel electrophoresis (PAGE) gels by their fluorescent label (Figure 2B,D).Together, these results show that both the antibody and the Cas9 precursors can be obtained in good purity and are capable of a high turnover in the protein trans-splicing reaction.Various chemical modifications could be introduced into either the IgG or the Cas9 proteins with the described reactions.
We further investigated whether the Cas9 part obtained from our bacterial protein production retained the ability to form the RNP complex with the sgRNA to introduce a double strand break in the targeted substrate DNA.For this, the control protein H 6 -SUMO-Cas9-SBP was prepared, which is similar to precursor 4 but lacks the Int C fragment.To reconstitute the RNP complex the protein was loaded with sgRNA according to standard protocols. 36Subsequently, a circular plasmid DNA containing the programmed cleavage sequence was added.Figure S3 shows that the plasmid DNA was cleaved in the nuclease assay and converted from the circular into the linear form, indicating successful RNP complex assembly.This cleavage reaction was dependent on the loading with sgRNA as in control experiments, in which the sgRNA was omitted, and no cleavage was observed.Importantly, similar results were obtained for the abovedescribed N-terminally 5( 6 protein, the product of the reaction between precursors 4 and 6 (Figure S2).Thus, the RNP complex could be reconstituted from the spliced Cas9 part and exhibited sgRNA-dependent nuclease activity.

| Split-intein mediated ligation of IgGs with Cas9
With the catalytically active components at hand, we next attempted the preparation of IgG-Cas9 conjugates as the central objective of this project (Figure 3A).To this end, we incubated the precursor proteins anti-CD33-Int N (3; at 3 μM) with H 6 -SUMO-Int C -Cas9-SBP (4; at 2 μM) at low temperatures (8 C) to provide maximal preservation of protein stability during the reaction.Of note, neither during sample preparation of 3 nor during the splice reaction were any reducing agents included to preserve the antibody disulfide bonds.The SDS-PAGE analysis of the protein trans-splicing reaction showed that after 8 h the reaction proceeded to near consumption of the Int C -Cas9 precursor 4, in agreement with its limited molarity and suggesting a virtually complete turnover.The protein was converted into a protein migrating at >200 kDa, which corresponds well with the calculated size of the desired splice product of the antibodies HC with Cas9 (MW of 220.8 kDa calculated for the HC-Cas9-SBP polypeptide chain).Thus, these results showed a remarkably efficient protein trans-splicing reaction to ligate the two very large IgG and Cas9 proteins.
We then reconstituted the RNP complex of the spliced anti-CD33-Cas9 conjugate by adding sgRNA and subsequently investigated the nuclease activity.Figure 4 shows DNA cleavage activity in the nuclease assay, suggesting the formation of an active RNP complex also in the IgG-Cas9 conjugate.
Finally, similar protein trans-splicing and nuclease assays were performed with the cixutumumab-Int N (1) and teprotumumab-Int N (2) precursors (Figure S3).Although slightly less complete in the protein trans-splicing reaction also for these proteins very good yields were To reduce the size of the antibody part of the chimeric molecule, it would be conceivable to use smaller antibody-like proteins or antibody-fragments like scFv or single-domain antibody-fragments (nanobodies or VHH). 37However, these proteins bind only monovalently and do not stimulate endocytosis after binding.In contrast, IgGs are bivalent and many are established for enhanced cellular uptake after binding due to increased endocytosis through triggering receptor dimerization or oligomerization by their crosslinking ability. 38,39For these reasons, IgGs appear more promising and more generally applicable.
Several milestones have to be mastered for an antibody-mediated delivery of the CRISPR/Cas complex to cells.The first is to conjugate the very large IgG protein to the Cas9 protein, which also is of considerable size.This milestone is accomplished in the present work using IgG HC can be equipped with one Cas9 despite its enormous size and the distance of the HC's C termini of only about 20 Å. 40 The IgGs employed in this study and their expression in CHO-cells are wellestablished and their cell-targeting and internalization ability has been demonstrated in previous work. 13,14 the next future steps, binding to cell surface epitopes and cellular uptake of the IgG-RNP conjugates has to be investigated.Furthermore, the RNP complex has to be cleaved from the IgG, and be released to the cytosol, reminiscent to the cargo in ADCs.The Cas9 constructs described in this study already contained an endosomal cleavage site between the IgG HC and the RNP for this purpose, that is, for cathepsin B cleavage in the late endosome. 41Likewise, an NLS sequence to effect translocation of the released RNP into the nucleus, where gene editing is to occur, was incorporated in our constructs.To The initial purification step was conducted via Ni-NTA gravity flow affinity chromatography of His-tagged proteins.Purification was performed at 4 C using flow columns with a bed volume of 1 ml of Ni-NTA resin (Cube Biotech).Two steps of washing, first with Ni-NTA buffer and with Ni-NTA buffer + 40 mM imidazole, were performed.Proteins were eluted in a single fraction (3 ml) with Ni-NTA buffer + 250 mM imidazole.The second step was the purification via streptactin gravity flow affinity chromatography of streptavidin binding peptide (SBP)-tagged proteins.The supernatant was passed through the pre-equilibrated column.The immobilized protein was washed with a 20-fold column volume of buffer W (100 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0).The bound protein was then eluted with buffer E (buffer W with 2.5 mM desthiobiotin).
fluorescein-CL NThe peptide containing the Int N fragment (with a Met15 to norleucine substitution) was assembled on TGR resin with a freshly coupled rink amide linker, by stepwise microwave assisted Fmoc-SPPS on a Liberty blue peptide synthesizer, operating on a 0.1-mmol scale.Activation of entering Fmoc-protected amino acids (Carbolution, Merck Millipore or Iris Biotech) was performed using Oxyma and DIC in DMF (1:1 molar ratio), with a four equivalent excess over the initial resin loading.Coupling steps were performed for initial 15 s at 75 C and 150 W followed by 110 s at 90 C and 30 W. Fmoc-deprotection steps were performed by treatment of the resin with a 20% piperidine solution in DMF for initial 15 s at 75 C and 150 W followed by 50 s at 90 C and 30 W. Following each deprotection step, the resin was washed thoroughly with DMF.5(6)-Carboxyfluorescein was manually coupled to the peptide by adding a solution of 5(6)-carboxyfluorescein-OH (2 eq.) (Sigma Aldrich), DIC (2 eq.), and HOAt (2 eq.) in DMF to the resin and shaking at room temperature for 16 h.Once the sequence assembly was finished, the resin was subsequently washed with DMF and DCM, and dried under nitrogen flow.The labeled peptide was finally cleaved off the resin by treatment with an ice-cold TFA, TIS, and water mixture (90:5:5) and allowed to shake at room temperature for 3 h, followed by purification by RP-HPLC.Analysis by ESI-mass spectrometry confirmed the sequence of the peptide: M(obs) = 3736.05Da; M(calc) = 3736.69Da.

( 5 -
mM) and TCEP (100 μM) for 20 min at 37 C.The fluorophoremaleimide reagent (Alexa647-C2 maleimide or fluorescein-5 maleimide, Thermo Fisher Scientific) was added at a final concentration of 200 μM and the reaction mixture was incubated for 2 h at 25 C.The reaction was quenched with DTT (2 mM) and the unreacted fluorophore was removed by affinity chromatography on Ni-NTA resin.
37 C and samples of 20 μl were taken and quenched by the addition of 1 μl of 500 mM EDTA in ddH 2 O at pH 8. Probes were further processed by the incubation with proteinase K for 20 min at 37 C.The plasmid pBRAF was incubated for 30 min with the single cutter Nde I at 37 C.The linearized plasmid was purified via agarose gel extraction and stored in 1Â nuclease buffer at À20 C. The linearized version was used as a positive control for the RNP-mediated linearization of the target plasmid.

3 | RESULTS AND DISCUSSION 3 . 1 |
Strategic considerationsIgGs are composed of light chains (LC; $25 kDa) and HC ($50 kDa) linked by disulfide bonds and assembled into a homodimer (total size $150 kDa).The expression of a genetic fusion with Cas9 ($160 kDa) has not been reported yet, likely because correct biosynthetic assembly of a genetically fused IgG-Cas9 is difficult in a single expression host.Of note, antibodies are of eukaryotic origin and require expression through the secretory pathway for correct chain assembly, disulfide formation, and glycosylation.In contrast, the CRISPR-Cas system is an RNP complex of prokaryotic origin that requires nuclear localization in mammalians to perform gene editing.To this end, Cas9 requires fusion with a nuclear localization sequence (NLS).Antibodies are commonly expressed in mammalian cells, such as Chinese hamster ovary (CHO) cells, and Cas9 is best expressed in bacteria, such as E. coli.We failed to express any protein from a genetic fusion of a Cas9-NLS gene with the open reading frame encoding an IgG HC (not shown), likely due to the above reasons or the incompatibility of Cas9 translation and folding in the secretory pathway.Therefore, we envisioned a posttranslational assembly using split inteins.Following separate expression and purification of the IgG and Cas9-NLS parts as IntN )-carboxyfluorescein-labeled Cf-Cas9-SBP F I G U R E 2 Fluorescent labeling of Cas9 and IgG using protein trans-splicing.Reactions were performed at 25 C and pH 7. (A) Reaction scheme for C-terminal labeling of the antibody-intein precursor.The cixutumumab antibody consists of its light chain LC (1 LC , calc.24.7 kDa) and heavy chain HC-Int N (1 HC , calc.56.1 kDa).The complementary split intein precursor SBP-Int C -Cys (Cf) carries an extein peptide tag labeled with carboxyfluoresceine (Cf) (5, 20.3 kDa).The resulting splice product is HC-Cf (SP, calc.54.1 kDa).(B) Analysis of the reaction shown in (A) between 1 (3 μM) and 5 (9 μM) using a reducing Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel with corresponding UV-illumination scan.(C) Reaction scheme of the N-terminal labeling of the intein-Cas9 precursor.H 6 -SUMO-Int C -Cas9-SBP (4, calc.198.6 kDa) was mixed with the synthetic peptide Cf-Int N (6, calc.3.7 kDa) containing carboxylfluoresceine in its short extein tag.The splice product is Cf-Cas9-SBP (SP, calc.171.6 kDa).(D) Analysis of the reaction shown in (C) between 6 (9 μM) and 4 (3 μM) using a reducing Coomassie-stained SDS-PAGE gel with corresponding UV-illumination scan.
obtained.So far, providing the Int C -Cas9 precursor in limiting molar stoichiometry could theoretically have led to the preferred formation of an only singly spliced IgG homodimer due to potential sterical hindrance at the neighboring two carboxy termini of the HC.We therefore tested if both HC of an intact IgG dimer could splice with Cas9 and performed reactions in which the Int C -Cas9 precursor 4 was provided in molar excess.FigureS4shows that when incubated with cixutumumab-Int N (1) the HC-Int N precursor was completely consumed.This observation suggests that indeed both HC of the IgG could be ligated with Cas9 to give an IgG-Cas9 assembly of about 470 kDa.Furthermore along these lines, our SDS-PAGE analyses showed no detectable amounts of undesired N-or C-terminal cleavage products of the intein, which would have resulted in protein bands of an isolated HC of the IgG or the isolated Cas9 protein, respectively.Taken together, our results suggested the utility of the splitintein mediated approach as a generally applicable method to prepare IgG-Cas9 conjugates with high efficiency and that these can be loaded with sgRNA to give active CRISPR/Cas reagents.F I G U R E 3 Split-intein mediated ligation of anti-CD33-IgG and Cas9.(A) Reaction scheme involving the antibody precursor consisting of the two light chains LC (3 LC , calc.24.7 kDa) and two heavy chains HC-Int N (3 HC , calc.52.9 kDa) as well as the Cas9 precursor H 6 -SUMO-Int C -Cas9-SBP (4, calc.198.6 kDa).(B) Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis using a coomassie-stained gel of a splice assay as shown in (A).The two lanes shown were spliced together from the original gel (see Supporting information).The reaction was performed in a molar excess of 3:1 of 3 HC (3 μM of anti-CD33-Int N ) over 4 (2 μM) at 8 C in a HEPES-KCl buffer system at pH 7. SP, splice product.To achieve targeting of CRISPR/Cas reagents to specific cells, a selective interaction of the cargo consisting of the nuclease reagents with cell-specific molecular structures is required.Current methods like virus or nanoparticle-mediated delivery show some cell selectivity; however, they have not yet been programmed for specific cell types of choice.Coupling the CRISPR/Cas-cargo to a specific ligand can enable targeting to cells expressing the cognate receptor,11 but this strategy has not yet been realized in a general way.Inspired by the success of ADCs as cellular targeting devices for cargo of scalable size, we here investigated the preparation of an IgG-Cas9 and IgG-RNP conjugates that could form the basis to deliver the RNP complex with the sgRNA selectively to cells.Importantly, antibodies are available against various extracellular epitopes, including many tumor-associated antigens, thus targeting IgG-RNP conjugates to various cell types would be conceivable using the same general platform.
protein trans-splicing of split intein precursor fusion proteins with the C terminus of the antibody HC, expressed in mammalian cells (CHO), and the Cas9 protein, overproduced in E. coli.The Cas9 part was successfully converted into the active RNP complex by loading with sgRNA.We have used a cysteine-less split intein for joining the two protein components, such that the addition of reducing agents can be circumvented during all steps of the protocols to keep the antibody disulfides intact.Furthermore, the Int N of this CL-intein28 consists of only 26 amino acids (plus 5 neighboring extein residues that also serve as linker), which might minimize adverse effects of the intein fusion on expression and folding of the antibody.Our data shows nearly quantitative splicing efficiencies with either the IgG or the Cas9 component in molar excess are possible.This finding also shows that each address release of the RNP out of the endosomes, Rouet et al. in their pioneering work co-incubated the asialoglycoprotein receptortargeted RNP prior to its addition to cells with an endosomolytic peptide.11Alternatively, genetic fusion with an endosomal escape sequence42 would be conceivable.In summary, as the first milestone on the route towards IgGmediated cell-specific CRIPSR/Cas-delivery the herein presented protocol presents an efficient and general approach to prepare IgG-Cas9 conjugates.With variations of the protocol either the IgG or the Cas9 protein can be selectively chemically modified at their terminal ends.This work underlines the potential of inteins as ligation tools to tracelessly link proteins of large size and from different expression hosts.

F I G U R E 4
Reconstitution of the RNP complex from the anti-CD33-Cas9 conjugate.(A) Scheme of the nuclease reaction.(B) Analysis of nuclease activity.Shown are UV-scans of ethidium bromide-stained agarose gels.The splice product of the reaction shown in Figure 3 was incubated with sgRNA to reconstitute the RNP complex and subsequently incubated for 20 min with a circular plasmid containing the programmed cleavage sequence (right panel).Positive control reactions with a Cas9 construct and plasmid DNA linearized by Nde I digestion are shown in the left panel.