Efficient Intracellular Delivery of CRISPR-Cas Ribonucleoproteins

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Letter

Efficient intracellular delivery of CRISPR-Cas ribonucleoproteins through receptor mediated endocytosis Romain Rouet, and Daniel Christ ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00116 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

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ACS Chemical Biology

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Efficient intracellular delivery of CRISPR-Cas ribonucleoproteins

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through receptor mediated endocytosis

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Authors

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Romain Rouet1,2* and Daniel Christ1,2

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Affiliations

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1

Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

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2

The University of New South Wales Sydney, Faculty of Medicine, St Vincent's Clinical

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School, Darlinghurst, NSW, Australia.

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Correspondence

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Romain Rouet, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.

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Email: [email protected]

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Abstract

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We recently reported a new delivery system harnessing surface receptors for targeted uptake

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of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this

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purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the

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asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human

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cells expressing the receptor. However, detailed mechanistic insights had remained unknown

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and editing efficiency low. Here we investigate the mechanism of endosomal escape as

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mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or

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Cas12a ribonucleoprotein complexes with increased editing efficiency.

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Introduction

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CRISPR-Cas systems have transformed biology by enabling rapid gene editing or modulation,

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with broad application in basic research 1, 2 and medicine 3, 4. Optimizing delivery methods for

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the CRISPR-Cas gene editing components (Cas9 endonuclease and single-guide RNA, forming

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a ribonucleoprotein (RNP)) to ensure safe, specific and efficient delivery into cells have been

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the focus of many recent research activities 5-7. Current methods generally rely on either virus-

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encoded components or alternatively recombinant ribonucleoproteins 5. Among viral delivery

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platforms, adeno-associated virus and lentivirus are highly efficient at delivering their genetic

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material into mammalian cells, although exhibit limited cell tropism for specific targeting 8. In

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addition, such viral platforms pose serious risks of immunogenicity, the possibility of random

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genomic integration, and restrict the size of encoded transgenes (to around 4 to 6 kb)

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Recombinant RNPs, on the other hand, are generally not immunogenic, characterized by low

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levels of off-target editing activity and do not rely on potentially hazardous genomic integration

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5, 11.

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delivery platforms for cellular uptake have been developed

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including electroporation (nucleofection), cell penetrating peptides and nanoparticle

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encapsulation, are characterized by variable efficiencies and are generally not cell type specific

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5.

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We previously demonstrated that a recombinant Cas9 RNP with a single nuclear localization

45

signal (Cas9-1NLS), bearing a small molecule ligand (ASGRL) specific for the

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asialoglycoprotein receptor (ASGR), is taken up selectively by hepatocarcinoma cell lines

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expressing the receptor

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peptide ppTG21, genomic insertions or deletions (indels) could be detected in approximately

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5% of treated cells, as identified by targeted next generation sequencing (NGS, with no

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detectable indels background in the absence of ppTG21 peptide). However, to achieve this

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level of efficacy, the method relied on using relatively high amounts of RNP (250 pmol). In

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addition, the mechanism of endosomal escape remains poorly understood.

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We identified here a new Cas9 construct, bearing a mCherry and 3NLS tags, that increases the

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ASGRL-mediated indels in human hepatocarcinoma cells (HepG2) by 2.5-fold compared to

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the previously reported construct (Cas9-1NLS), while reducing the amount of RNP for

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incubation by 5-fold (only 50 pmol). While some background editing is observed with this new

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construct in HepG2, none is detectable in other cell lines. We then demonstrate that co-

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incubation of the endosomolytic peptide ppTG21 and the ASGRL-Cas9 RNP is necessary to

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observe gene editing. Using inhibitors of endocytosis pathways and ppTG21 peptide

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mutagenesis, we also demonstrate that endosomal escape of the RNP with ppTG21 is pH-

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dependent and mediated by histidine residues on the surface of ppTG21. Finally, we show that

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the described targeted delivery approach is applicable to the Cas12a system.

9, 10.

However, unlike viruses, RNPs cannot readily enter mammalian cells, and thus multiple

12.

12, 13.

These delivery platforms,

When administered in the presence of the small endosomolytic

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Results and Discussion

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Increased editing efficiency with ASGRL conjugated Cas9-mCherry-3NLS

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In order to further increase the efficacy of gene editing, we utilized a high-throughput method

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based on T7 Endonuclease I DNA mismatch detection (T7E1 assay) 2


14-16.

We first evaluated

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the editing efficiency of the ASGRL-Cas9-1NLS RNP (250 pmol or 333 nM) used in our prior

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study by co-incubation with ppTG21 endosomolytic peptide (7800 pmol or 10 µM) with the

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hepatocarcinoma HepG2 cell line. As previously observed, we detected only a small proportion

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of indels at the targeted EMX1 locus (1.2% compared to 5% when analyzed by NGS 12) (Supp.

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Fig. 1A), highlighting the limitations of the method for detection of low indel levels.

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We next attempted to identify a construct with higher editing efficiency. We had previously

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demonstrated that Cas9 RNP, bearing the ASGRL and a mCherry and 3NLS tags (ASGRL-

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Cas9-mCherry-3NLS; Fig. 1A and Supp. Fig. 1C), displays faster ASGR-mediated uptake

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than its 1NLS counterpart on HepG2 cells, although some level of non-specific uptake was

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also observed upon prolonged incubation 12. We sought to investigate whether this construct

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would also display higher editing levels. Indeed, we observed more than 3% editing efficiency

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using ASGRL-Cas9-mCherry-3NLS RNP using the T7E1 assay and the previously reported

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conditions (250 pmol of RNP and 7800 pmol of ppTG21) 12 (Supp. Fig. 1B).

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We next screened for assay conditions that further improved editing efficiency at lower RNP

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concentrations, by titrating ppTG21 in the presence of 50 pmol (66 nM) of ASGRL-Cas9-

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mCherry-3NLS RNP. Intriguingly, we observed increased gene editing compared to ASGRL-

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Cas9-1NLS, with up to 16% indels observed (Fig. 1B). Importantly, we retained the ASGR

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selectivity previously analyzed by live cell imaging

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editing efficiency observed in the presence of ASGRL-RNP compared to RNP only. Moreover,

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the high background gene editing with the RNP only was not observed when analyzed in a

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different hepatocarcinoma cell line (SkHep), lacking expression of the ASGR, or in HEK293

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cells (Supp. Fig. 2). Thus, we consider the background editing to be an artefact of the HepG2

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cell line with prolonged RNP incubation. To minimize non-ASGR mediated gene editing, we

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initially chose 750 pmol (1 µM) of ppTG21 for our experiments (Fig. 1B). Further screening

92

of RNP concentrations did not yield further improvements (Supp. Fig. 3). However, our results

93

suggest that the ppTG21 to RNP ratio is key to successful endosomal escape, with optimal

94

ratios between 15:1 and 30:1 (Supp. Fig. 3). This observation is in excellent agreement with

95

our previously reported finding for Cas9-1NLS complexes (~30:1 ratio, with 250 pmol or 333

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nM of RNP and 7800 pmol or 10 µM of ppTG21) 12.

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Based on the optimizations carried out here, the following assay conditions were used for all

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subsequent experiments: 50 pmol (66 nM) of RNP and 750 pmol (1 µM) of ppTG21, which

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achieved gene levels of 9% for the ASGRL RNP and 4% for the background RNP (devoid of

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the ASGRL) in HepG2 cells using the T7E1 assay (Fig. 1C). These results were confirmed by

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targeted NGS, with approximately 13% editing efficiency observed for the ASGRL-Cas9-

12,

with approximately 2.5-fold higher


3


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mCherry-3NLS compound, with background activities observed at 6% for the Cas9-mCherry-

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3NLS and 0% for both ASGRL- and Cas9-mCherry-3NLS in the absence of ppTG21 (Supp.

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Table 1 and Methods). These editing efficiencies were achieved using considerably lower

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amount of RNP (5-fold less) and ppTG21 peptide (10-fold less) than reported in our previous

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study 12. In fact, the 50 pmol RNP amount used in the optimized protocol developed here is the

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same as used in nucleofection. Nucleofection is currently the gold standard in terms of editing

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efficiency for RNP mediated genome editing in human cells 5. It thereby allows direct gene

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editing efficiency comparisons between the two delivery technologies. Importantly, non

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ASGR-mediated RNP uptake is not concerning as gene editing occurs only in the presence of

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the ppTG21 peptide (Fig. 1B-C). Therefore, the mechanism of RNP endosomal escape must

112

be similar, regardless of the uptake pathway.

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Simultaneous incubation of ASGRL-Cas9 RNP and ppTG21 is necessary for endosomal

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escape

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In order to confirm the requirement for co-incubation of the ASGRL-Cas9 RNP (Cas9

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comprising mCherry and 3NLS tags) and the endosomolytic peptide ppTG21 to achieve

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genome editing, we performed pulse/chase experiments. We incubated HepG2 cells with

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ASGRL-Cas9 RNP for 3 h, then washed the cells with heparin to remove any membrane bound

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RNP and subsequently incubated with ppTG21 for a further 40 h. Interestingly, no gene editing

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was observed under these conditions (Supp. Fig. 4A). A similar assay where the ASGRL-Cas9

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RNP and ppTG21 incubations were inversed also did not yield any gene editing (Supp. Fig.

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4A). This demonstrates that co-incubation of the RNP and the peptide is required for intra-

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cellular endosomal escape and suggests that the peptide is being internalized together with the

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RNP via ASGR-mediated or non-specific endocytosis pathways. To further demonstrate the

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requirement for simultaneous incubation of the two components, we co-incubated HepG2 cells

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with the RNP and ppTG21 for only 3 h, then washed the cells and incubated them with media

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for a further 40 h. This resulted in 2.6% of indels for the ASGRL-Cas9 RNP and only 0.9% for

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the background Cas9 RNP, representing a 3-fold ASGR-specific increase in editing, and

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correlated with the previous live cell imaging study (3-fold increase after 4 h) 12 (Supp. Fig.

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4B).

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ASGRL-Cas9 RNP is rapidly degraded upon receptor-mediated uptake

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We next sought to estimate how rapidly endosomal escape occurs by monitoring intracellular

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RNP degradation. It was previously demonstrated that the ASGR pathway directs internalized

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glycosylated protein directly to the lysosome 17. We therefore monitored the intracellular levels

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of RNP over time in HepG2 cells. Cells were incubated with 100 pmol (132 nM) of ASGRL4


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Cas9 RNP (to allow for better quantification compared to 50 pmol) and at different time points

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washed with heparin, harvested and total RNA extracted. We intentionally avoided using

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ppTG21 in this assay to allow the RNP to migrate through the ASGR endocytic pathway.

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Levels of Cas9 RNP were determined using RT-qPCR to detect the sgRNA and compared to

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endogenous house-keeping genes and a spike-in control of 100 pmol RNP for quantification.

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Overall, we observed a sharp increase in RNP accumulation for the first 2 h, reaching up to

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about 25 pmol (25% of total RNP); however, a slow decrease in RNP level occurred soon after,

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dropping by 2.5-fold after 4 h (10 pmol - 40% of endocytosed RNP) and 25-fold after 8 h (1

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pmol - 4% of endocytosed RNP), which may correspond to lysosomal degradation (Fig. 2).

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We previously demonstrated a linear uptake of the ASGRL-Cas9-mCherry-3NLS RNP in

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HepG2 cells for the first 12h following incubation 12, suggesting that lysosomal degradation

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occurs rapidly. Overall, the results indicate that ppTG21-dependant RNP endosomal escape

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may occur rapidly, most likely in the first 8h following uptake. It also indicates that a rapid

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uptake may be necessary to achieve sufficient ppTG21 concentration in the endosome and thus

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induce RNP escape, thereby potentially explaining the lower editing efficiency with the 1NLS

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RNP.

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Histidine residues on the surface of ppTG21 allow escape of the ASGRL-Cas9 RNP from

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the late endosome

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To understand the mechanism of endosomal escape mediated by the ppTG21 peptide, we first

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performed alanine scanning mutagenesis of surface exposed residues and evaluated the mutant

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peptides for gene editing by co-incubation with ASGRL-Cas9 RNP in HepG2 cells. We

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observed a reduction in indel levels, between 30% and 80%, when single residues were mutated

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to alanine (Fig. 3A). In particular, histidine residues at positions 11 and 19 and serine residue

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at position 12 show the most dramatic decrease in gene editing (70-80%). To confirm the

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importance of histidine residues in endosomal escape capability of ppTG21, we evaluated

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mutant peptides with two histidine substitutions to alanine. Due to the increased

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hydrophobicity of the peptides, only three peptides could be synthesized: 8/15AA, 8/19AA and

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11/19AA. All three peptides dramatically reduced indel levels by 85 to 95%, thus confirming

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the importance of histidine residues for escape from endocytic pathways (Fig. 3A). The results

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are in agreement with previous findings showing that peptide variants of ppTG21, with

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substitution of all histidine residues to glutamic acid (JTS-1) or lysine (ppTG1), were not

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capable of endosomal escape 12, 18 (Fig. 3A). Interestingly, when we co-incubated the ASGRL-

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Cas9 RNP in HepG2 cells with previously described histidine-rich endosomolytic peptides

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such as H5WYG 19, LAH4 20, EB1 21, or the small chemical drug chloroquine 22, we did not


5


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observe gene editing. This suggests a novel mechanism of endosomal escape using the ppTG21

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peptide.

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We finally decided to map the localization of the escape along the endocytic pathway. To

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achieve this, we performed the same ASGRL-Cas9 RNP ppTG21 co-incubation experiments,

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with HepG2 cells, this time the cells being treated with chemical inhibitors of endocytosis 23.

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We used the following inhibitors: dynasore, a dynamin inhibitor that will arrest ASGR clathrin-

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mediated endocytosis on the cell surface; N-ethyl-isopropyl amiloride (EIPA), an actin

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remodeling inhibitor; wortmannin, a PI3K inhibitor involved in phagocytosis pathway;

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bafilomycin A, a V-type ATPase inhibitor preventing endosome maturation to late endosome

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and lysosome. EGTA, a calcium ion chelator, preventing ASGRL from binding to its receptor,

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and asialo-orosomucoid (ASOR), an ASGRL competitor for its receptor, were used as controls

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17, 24.

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endocytic pathways without being toxic for cells 23. HepG2 cells were initially pre-incubated

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with the inhibitors for 1 h, then the ASGRL-Cas9 RNP and ppTG21 added to allow for

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immediate pathway inhibition upon RNP uptake. Cells were incubated with the inhibitors and

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RNP/ppTG21 for only 24 h (instead of 48 h), to mitigate loss of inhibition over prolonged

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incubation. We observed that all inhibitors decreased gene editing levels to various degrees

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(Fig. 3B). More specifically, editing was reduced by over 90% with EGTA and ASOR, and

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70% with dynasore, thus confirming the predominant ASGR dynamin-mediated RNP uptake

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but also the presence of minor alternative uptake pathways. Almost complete inhibition with

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EIPA (preventing actin remodeling) also confirmed RNP entry via endocytic pathways and not

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through pore formation. Weak inhibition (~ 30%) by wortmannin suggested that this may

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represent the alternative RNP uptake pathway. Indeed, it was recently demonstrated that

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wortmannin blocks micropinocytosis, a non-specific uptake pathway, of nanoparticles in

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HepG2 25. More striking was the complete loss of gene editing in the presence of bafilomycin

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A. This suggests that inhibition of endosome maturation, from early to late stage, abrogates

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RNP endosomal escape and thus implies that escape may be occurring in the late endosome.

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Endosome maturation is characterized by acidification of the endosomal milieu, therefore

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suggesting a pH-dependent escape mechanism of the RNP with the ppTG21 peptide. Combined

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with the mutagenesis results on the ppTG21 peptide, this suggests that protonation of histidine

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residues, during the maturation of endosomal vesicles, leads to activation of the endosomolytic

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property of the peptide.

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ppTG21 mediates escape of ASGRL conjugated Cas12a

6

All the inhibitors were used at concentrations that were previously shown to inhibit


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We finally investigated whether Acidaminococcus sp. Cas12a could also be harnessed for

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receptor-mediated gene editing using ppTG21 as endosomolytic peptide. Cas12a was shown

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to be an RNA-guided DNA endonuclease 26 and active in eukaryotic organisms

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Cas9, Cas12a recognizes a different PAM sequence (5’-TTTV), generates staggered double

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stranded DNA breaks and is capable of processing its own pre-crRNA 29.

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Following analogous methodology to Sp. Cas9 12, As. Cas12a (bearing two C-terminal NLS

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tags) was conjugated with pyridyl-disulphide ASGRL onto surface exposed cysteine residues.

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To avoid over-labeling of the protein (Cas12a contains 8 cysteine residues) and thus possibly

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affect endonuclease or crRNA processing activities, we used limited conjugation with only 2-

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fold molar excess of ASGRL compared to Cas12a. Native and tryptic digestion mass

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spectrometry revealed that the ASGRL-Cas12a contained a mixture of single- and bis-

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conjugated proteins, with only cysteine residue as positions 334, 379 and 1248 being labeled

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with an ASGRL (Supp. Fig. 5A). In vitro pre-crRNA processing and DNA endonuclease

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activities remained unaffected by the presence of the conjugated-ASGRL (Fig. 5A-B).

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We next evaluated the gene editing activity of the Cas12a RNP. We used a pre-crRNA targeting

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human PCSK9 locus, assembled it with Cas12a to generate RNP and incubated with HepG2

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cells for 48 h. As previously observed with Cas9, no gene editing was observed in the absence

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of the endosomolytic peptide ppTG21 (Fig. 4C and Supp. Fig. 5B). However, co-incubation

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of 50 pmol of Cas12a RNP with only 375 pmol ppTG21 (7.5:1 molar ratio) successfully

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achieved gene editing to about 4.5% for the ASGRL-Cas12a RNP, and 2% for the background

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control Cas12a RNP. Titration of ppTG21 did not lead to improvement in gene editing levels

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(Supp. Fig. 6). Overall, this confirms that ppTG21 is a universal endosomolytic peptide that

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allows endocytosed CRISPR-Cas RNP, either through receptor-mediated endocytosis or non-

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specific uptake pathways such as macropinocytosis, to escape to the cytoplasm, enabling

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subsequent nuclear import for gene editing. The gene editing levels observed here are lower

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than those observed when the RNP is physically introduced in HepG2 cells by nucleofection

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(around 20%) but still substantial (Fig. 4C and Supp. Fig. 5B). Altogether, the results confirm

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that Cas12a construct optimization may also be required to improve receptor-mediated gene

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editing efficiencies, similar to what was performed for Cas9, with attachment of various tags.

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Discussion

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Our data highlights key mechanisms of CRISPR-Cas endonuclease RNP endosomal escape

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employing co-incubation of the cell-specific targeting RNP with the small endosomolytic

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peptide ppTG21. In particular, it was demonstrated that the RNP and the peptide must be co-

236

incubated to allow endosomal escape, as sequential incubations do not yield any gene editing.


27, 28.

Unlike

7


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In addition, inhibition of actin remodeling almost completely prevented gene editing, thus

238

ruling out non-specific entry via pore formation on the cell membrane. Gene editing was also

239

abolished in the presence of bafilomycin A, which prevents maturation of the early endosome

240

to the late endosome and lysosome, thus suggesting that endosomal escape occurs at the late

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endosome stage, before any protein degradation occurs in the lysosome. Moreover,

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endosomolytic activity of ppTG21 was shown to be pH-dependent as substitutions of histidine

243

residues to alanine or charged amino acids (lysine or glutamic acid) reduced or abolished gene

244

editing capabilities. Altogether, this suggest a model where the ASGRL-RNP and the ppTG21

245

peptide are taken up together via ASGR-mediated endocytosis, and subsequently migrate along

246

the endocytic pathway. There, the intra-endosomal milieu progressively acidifies until it

247

reaches a point where ppTG21 allows disruption of the late endosome and facilitates escape of

248

the trapped RNP to the cytoplasm.

249

It is unclear why the Cas9 comprising the mCherry and 3NLS tags demonstrated superior gene

250

editing compared to the 1NLS tag counter-part. However, considering the fast RNP

251

degradation occurring in the absence of ppTG21 peptide, one may suggest that fast uptake is

252

necessary to achieve sufficient ppTG21 concentration in the late endosome and subsequent

253

escape. The slower uptake of the Cas9-1NLS RNP may not be enough to compensate for the

254

fast degradation, thereby allowing limited amount of RNP to escape 12.

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Better understanding this mechanism will allow for the engineering of peptides with improved

256

endosomolytic properties. Indeed, the difference in gene editing levels between nucleofection

257

and receptor-mediated uptake (about 2-fold for Cas9-mCherry-3NLS and 5-fold for Cas12-

258

2NLS) and the previously demonstrated rapid and strong ASGR-mediated uptake by live cell

259

imaging

260

process. Further improvements may lead to gene editing efficiencies that may surpass that of

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the current gold standard for CRISPR-Cas RNP delivery: nucleofection.

12

suggest that endosomal escape with ppTG21 is currently a relatively inefficient

262 263

Methods

264

Cell culture and reagents

265

Human HepG2 and SkHep hepatocarcinoma cell lines and HEK293 cell line were obtained

266

from ATCC and cultivated in EMEM media (ATCC) or DMEM media for HEK293 (Gibco)

267

supplemented with 10% FBS and penicillin/streptomycin at 37C with 5% CO2.

268

Endosomolytic and cell-penetrating peptides (lyophilized) were purchased from Anaspec and

269

reconstituted in DMSO at high concentration (10 mM). Heparin, EGTA, Dynasore, EIPA,

8


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Wortmannin and Bafilomycin A were purchased from Sigma. Pyridyl-disulphide ASGRL, and

271

ASOR (asialo-orosomucoid) was supplied by Pfizer.

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Protein expression, purification, sgRNA/pre-crRNA synthesis and RNP formation

273

Cas9 and Cas12a proteins were expressed and purified as previously described 12. Single-guide

274

RNA and pre-crRNA were synthesized by T7 in vitro transcription, purified by PAGE and

275

refolded in 20 mM HEPES pH 7.5, 1 mM MgCl2, 150 mM KCl, 10% glycerol as previously

276

described 16. Pre-crRNA was synthesized with a self-cleaving hepatitis delta virus ribozyme at

277

its 3’-end to ensure homogeneous blunt ends. Pre-crRNA DNA template was generated using

278

Oligos 1 and 2 at 1 µM and Fwd 1 and Rev 1 at 10 µM as previously described 16 (Supp. Table

279

2). RNP was assembled by incubating Cas9 and sgRNA or Cas12a and pre-crRNA at a ratio

280

of 1:1.2 at 37C for 10 min.

281

Cas protein ASGRL conjugation

282

Pyridyl-disulfide ASGRL was dissolved to a concentration of 8 mM in DMSO. Cas9 or Cas12a

283

were filtered through 0.2 µm filter units prior to conjugation. Conjugation was carried out using

284

a 20:1 molar ratio of ASGRL (diluted 10-fold in 20 mM HEPES pH 7.5, 150 mM KCl, 10%

285

glycerol) to Cas9 or 2:1 for Cas12a at 4C over-night. The conjugated protein was further

286

purified by size-exclusion chromatography in 20 mM HEPES pH 7.5, 150 mM KCl, 10%

287

glycerol to remove unreacted ASGRL. Conjugation was assessed by native mass spectrometry

288

as previously described 12. Tryptic digestion was performed by denaturation in 6 M urea and

289

incubation at 55C for 20 min and over-night digestion at room temperature with mass spec

290

grade trypsin (Promega) after dilution in 50 mM Tris pH 7.0, 1 mM CaCl2 and ~ 0.5 M urea

291

final concentration. Digested fragments were analyzed by mass spectrometry.

292

Cell-based assays and T7E1 assay

293

For nucleofection assay, cells were harvested from culture plates, washed once with PBS and

294

resuspended at 10,000,000 cells/ml in SF buffer (Lonza). Ten microliters of Cas9 or Cas12a

295

RNP (containing the desired amount of RNP) were added to 20 µl of cells in SF buffer and

296

transferred to wells of a nucleofection plate (Lonza). Cells were electroporated using the 96-

297

well shuttle nucleofector (Lonza) and the HepG2 settings, incubated at room temperature for

298

10 min, resuspended in growth media and finally transferred onto a 12-well culture plate (1

299

ml/well).

300

For receptor-mediated uptake assay, cells were harvested, seeded in 24-well plates at 80,000

301

cells/well (700 µL) and incubated at 37C for 4-12 h. RNP was diluted into 50 µl of Opti-MEM


9


302

and incubated for 5 min. Endosomolytic peptide was then dispensed to the Cas9 RNP/Opti-

303

MEM mix, incubated for 5 min and dispensed to cells.

304

Cells were harvested in Quick Extract buffer (Epicenter) after 48 h co-incubation with RNP

305

and peptide (unless stated otherwise), incubated for 10 min at 65C then for 10 min at 95C.

306

Genomic DNA concentrations were estimated by measuring absorbance at 260 nm. Targeted

307

locus was amplified by PCR (Kapa Biosystems) and quantified on agarose gel stained with

308

SYBR Gold (Invitrogen) by comparison to a standard. 150 ng of PCR product was melted then

309

hybridized and subjected to cleavage with T7 Endonuclease I (NEB). The resulting reaction

310

was run on a 2% agarose gel stained with SYBR Gold and the cleavage bands quantified using

311

Image Lab (BioRad). The fraction of cleaved PCR product (f(cut)) was determined by the ratio

312

of cleaved DNA (intensity of cleaved bands) to total DNA (combined intensity of all bands).

313

The percentage of indels was determined as follows: 𝑖𝑛𝑑𝑒𝑙𝑠 (%) = 100 × (1 ― √(1 ― 𝑓(𝑐𝑢𝑡)).

314

Genomic DNA – Cas9 target sequences (PAM):

315

Human EMX1 exon 1: GTCACCTCCAATGACTAGGG (TGG)

316

Genomic DNA – Cas12a target sequences (PAM):

317

Human PCSK9 exon 4: (TTTC) CCGGTGGTCACTCTGTATGCTGG

318

Targeted next generation sequencing

319

Genomic DNA was amplified by PCR (Kapa Biosystems) with primers Fwd 2 and Rev 2

320

(Supp. Table 2) comprising a unique molecular identifier (N10) and stub sequence (5’-

321

GCTCTTCCGATCT - 3’) and purified using QIAquick PCR purification kit (Qiagen). Indexes

322

were subsequently added by PCR and the final libraries ran on an agarose gel and extracted.

323

Libraries were quantified by Qubit (Thermo Fisher), pooled and sequenced on a HiSeq 2500

324

(Illumina) using 250 bp paired-end sequencing. Paired-end reads were first merged using

325

PEAR 30, then unique molecular identifier duplicates were removed using PRINSEQ 31, and

326

finally indels were analyzed using Cas-Analyzer tool 32.

327

RT-qPCR

328

HepG2 cells were seeded in individual 3cm petri dishes at a density of 100,000 cells/well. 100

329

pmol of ASGRL-Cas9 RNP were dispensed onto cells and incubated for different times at 37C

330

with 5% CO2. Cells were washed once with PBS and twice with heparin (200 U/ml in PBS),

331

and lysed in Trizol (Life Technologies). Total RNA was extracted from the Trizol, treated with

332

DNase I (NEB) and purified by phenol/chloroform extraction. As a control, 100 pmol of RNP

333

was spiked into Trizol lysed untreated cells. cDNA was generated using 1 µg of RNA and the

334

Superscript Vilo kit (Invitrogen), spiked with sgRNA Rev primer (Supp. Table 2). qPCR was

10


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335

performed using the Dynamo SYBR green mix (Thermo Scientific) and the relative quantities

336

of sgRNA determined by double ΔCT analysis between sgRNA (using Fwd 3 and Rev 3) and

337

GAPDH (using Fwd 4 and Rev 4) house-keeping gene PCR (sequences in Supp. Table 2).

338

Cas12a in vitro pre-crRNA processing and DNA cleavage

339

For processing assay, in vitro transcribed pre-crRNA at 20 µM was mixed with Cas12a at 20

340

µM in 20 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM CaCl2, 150 mM KCl and incubated at

341

37C for 15 min followed by 95C for 5 min. Half of the reaction was ran on a PAGE gel and

342

visualized by staining with SYBR Gold. 0.4 pmol (about 150 ng) of human PCSK9 PCR

343

product (amplified using Fwd 5 and Rev 5 primers; sequences in Supp. Table 2) was mixed

344

with 4 pmol of RNP (in 20 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM CaCl2, 150 mM KCl)

345

and incubated at 37C for 30 min followed by 95C for 5 min. Cleavage products were

346

visualized on agarose gel stained with SYBR Gold.

347 348

References

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Zhang, F. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system, Cell 163, 759-771. 27. Moreno-Mateos, M. A., Fernandez, J. P., Rouet, R., Vejnar, C. E., Lane, M. A., Mis, E., Khokha, M. K., Doudna, J. A., and Giraldez, A. J. (2017) CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing, Nat Commun 8, 2024. 28. Kim, Y., Cheong, S. A., Lee, J. G., Lee, S. W., Lee, M. S., Baek, I. J., and Sung, Y. H. (2016) Generation of knockout mice by Cpf1-mediated gene targeting, Nat Biotechnol 34, 808-810. 29. Fonfara, I., Richter, H., Bratovic, M., Le Rhun, A., and Charpentier, E. (2016) The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA, Nature 532, 517-521. 30. Zhang, J., Kobert, K., Flouri, T., and Stamatakis, A. (2014) PEAR: a fast and accurate Illumina Paired-End reAd mergeR, Bioinformatics 30, 614-620. 31. Schmieder, R., and Edwards, R. (2011) Quality control and preprocessing of metagenomic datasets, Bioinformatics 27, 863-864. 32. Park, J., Lim, K., Kim, J. S., and Bae, S. (2017) Cas-analyzer: an online tool for assessing genome editing results using NGS data, Bioinformatics 33, 286-288.

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Acknowledgments

449

R.R. thanks J. Doudna, C. Fellmann and members of the Doudna lab at UC Berkeley for helpful

450

discussions. R.R. was a member of the Doudna lab (2015-2017) as part of his early career

451

postdoctoral fellowship from the Australian National Health and Medical Research Council.

452

R.R. acknowledges support from the NHMRC (APP1090875). R.R. thanks members of Pfizer

453

for reagents.

454 455

Author contributions

456

RR designed and performed research; RR analyzed data; RR & DC wrote the manuscript.

457 458

Competing interests

459

The authors declare that they have no competing interests.

460 461

Data availability

462

All data generated or analyzed during this study are included in this published article (and its

463

Supporting Information files).

464 465 466 467 468

Nomenclature CRISPR Clustered Regularly Interspaced Short Palindromic Repeats RNP RiboNucleoProtein ASGR ASialoGlycoprotein Receptor


13


469 470 471 472 473 474

ASGRL NLS T7E1 ASOR EIPA PAGE

14

ASialoGlycoprotein Receptor Ligand Nuclear Localization Signal T7 Endonuclease I ASialo-ORosomucoid N-ethyl-isopropyl amiloride PolyAcrylamide Gel Electrophoresis


Page 14 of 21

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475


Figure legends

476 477

Figure 1. Editing efficiency of Cas9-mCherry-3NLS RNP in HepG2 cells. (A) Cartoons

478

representing the previous construct Cas9-1NLS and new one Cas9-mCherry-3NLS (Cas9 in

479

blue, ASGRL in pink, NLS in yellow and mCherry in red). (B) Comparison of human EMX1

480

gene editing efficiency between ASGRL-Cas9-mCherry-3NLS RNP and Cas9-mCherry-3NLS

481

RNP upon co-incubation with ppTG21 endosomolytic peptide. 50 pmol of RNP was incubated

482

with HepG2 cells for 48h with an increasing amount of ppTG21 (0, 50, 200, 800 and 1600

483

pmol or 0, 66, 266, 1067, 2133 nM) and indels analyzed by T7E1 assay (shown in one

484

representative of n=2 independent experiments). (C) Validation of gene editing efficiency with

485

Cas9-mCherry-3NLS RNP only in the presence of ppTG21 or by nucleofection. 50 pmol of

486

RNP was either incubated with or without 750 pmol of ppTG21 or nucleofected in HepG2 cells

487

for 48h and indels analyzed by T7E1 assay (n=6 independent experiments; unpaired t-test).

488 489

Figure 2. Quantification of ASGRL-Cas9 RNP half-life in HepG2 cells upon receptor-

490

mediated endocytosis. 100 pmol (132 nM) of ASGRL-Cas9 RNP was incubated with HepG2

491

cells and the cells washed and harvested at different time points. Total RNA was extracted, and

492

RT-qPCR performed on sgRNA and GAPDH house-keeping gene. Amount of sgRNA was

493

compared to a spike-in control of 100 pmol (n=2 independent experiments).

494 495

Figure 3. Mechanism and localization of endosomal escape with ppTG21 peptide. (A)

496

Effect of substitutions of surface exposed residues to alanine on the editing efficiency of the

497

peptide. 50 pmol (66 nM) of ASGRL-Cas9 RNP was incubated with HepG2 cells and with 750

498

pmol (1 µM) of peptide for 48h and indels analyzed by T7E1 assay. Single mutations are shown

499

in blue and double mutations in purple (n=3 independent experiments). (B) Effect of endocytic

500

pathway inhibitors on the editing efficiency of the Cas9 RNP co-incubated with ppTG21

501

peptide. HepG2 cells were pre-incubated with EGTA at 0.5 mM, ASOR at 80 µM, dynasore at

502

80 µM, EIPA at 50 µM, bafilomycin A at 200 nM, or wortmannin at 200 nM for 1h before the

503

RNP and ppTG21 were added. Cells were harvested after 24h incubation and indels analyzed

504

by T7E1 assay (n=3 independent experiments).

505 506

Figure 4. PCSK9 gene editing with As Cas12a RNP and ppTG21. (A) In vitro pre-crRNA

507

processing with ASGRL-Cas12a and Cas12. Cas12a at 20 µM and pre-crRNA at 20 µM were

508

mixed, incubated at 37C for 15 min followed by 95C for 5 min and resolved on PAGE gel


15


509

(shown is one representative of n=2 independent experiments). (B) In vitro DNA cleavage with

510

ASGRL-Cas12a and Cas12a RNP. 200 ng (~ 0.4 pmol) of PCSK9 PCR product was incubated

511

with 2 pmol of RNP at 37C for 30 min followed by 95C for 5 min and resolved on agarose

512

gel (shown is one representative of n=2 independent experiments). (C) Validation of gene

513

editing efficiency with Cas12a RNP only in the presence of ppTG21 or by nucleofection. 50

514

pmol (66 nM) of RNP was either incubated with or without 375 pmol (500 nM) of ppTG21 or

515

nucleofected in HepG2 cells for 48h and indels analyzed by T7E1 assay (n=4 independent

516

experiments except for nucleofection where n=2, unpaired t-test).

517

16


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518 519


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Figure 2

520 521

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Figure 3

A 1 5 10 15 20 (ppTG21) GLFHALLHLLHSLWHLLLHA

GLFAALLHLLHSLWHLLLHA GLFHALLALLHSLWHLLLHA GLFHALLHLLASLWHLLLHA GLFHALLHLLHALWHLLLHA GLFHALLHLLHSLAHLLLHA GLFHALLHLLHSLWALLLHA GLFHALLHLLHSLWHLLL AA GLFHALLALLHSLWALLLHA GLFHALLALLHSLWHLLLAA GLFHALLHLLASLWHLLLAA (JTS-1) GLFEALLELLESLWELLLEA (ppTG1) GLFKALLKLLKSLWKLLLKA

B

522 523


19


524

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80x40mm (300 x 300 DPI)


Efficient Intracellular Delivery of CRISPR-Cas Ribonucleoproteins

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