Genome-editing Proteins Research Articles

Efficient genome editing by controlled release of Cas9 ribonucleoprotein in plant cytosol using polymer-modified microneedle array

Direct delivery of genome-editing proteins into plant tissues could be useful in obtaining DNA-free genome-edited crops obviating the need for backcrossing to remove vector-derived DNA from the host genome as in the case of genetically modified organisms generated using DNA vector. Previously, we successfully delivered Cas9 ribonucleoprotein (RNP) into plant tissue by inserting microneedle array (MNA) physisorbed with Cas9 RNPs. Here, to enhance protein delivery and improve genome-editing efficiency, we introduced a bioactive polymer DMA/HPA/NHS modification to the MNA, which allowed strong bonding between the proteins and MNA. Compared with other modifying agents, this MNA modification resulted in better release of immobilized protein in a plant cytosol-mimicking environment. The delivery of Cas9 RNPs in Arabidopsis thaliana reporter plants was improved from 4 out of 17 leaf tissues when using unmodified MNAs to 9 out of 17 when using the polymer-modified MNAs. Further improvements in delivery efficiency can be envisaged by optimizing the polymer modification conditions, which could have significant implications for the development of more effective plant genome editing techniques.

MRNA-based cancer therapeutics.

Due to the fact that mRNA technology allows the production of diverse vaccines and treatments in a shorter time frame and with reduced expense compared to conventional approaches,there has been a surge in the use of mRNA-based therapeutics in recent years. With the aimof encoding tumour antigens for cancer vaccines, cytokines for immunotherapy, tumour suppressors to inhibit tumour development, chimeric antigen receptors for engineered T cell therapyor genome-editing proteins for gene therapy,many of these therapeutics have shown promising efficacy in preclinical studies, and some have evenentered clinical trials.Given the evidence supporting the effectiveness and safety of clinically approved mRNA vaccines, coupled with growing interest in mRNA-based therapeutics, mRNA technology is poised to become one of the major pillars in cancer drug development. In this Review, we present in vitro transcribed mRNA-based therapeutics for cancer treatment, including the characteristics of the various types of synthetic mRNA, the packaging systems for efficient mRNA delivery, preclinical and clinical studies, current challenges and future prospects in the field. We anticipate the translation of promising mRNA-based treatments into clinical applications, to ultimately benefit patients.

Improved genome editing by an engineered CRISPR-Cas12a.

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction

Plasmid mutagenesis is an essential step to engineer protein variants with desired properties. Direct generation of mutations on plasmids is a promising method compared to PCR-based methods and subcloning, yet it suffers from the sequence restriction in the generation of single-stranded circular plasmids. Here we demonstrate the CRISPR/Cas system-guided plasmid mutagenesis, a method using gRNA/Cas9 nickase complex to generate single-stranded circular plasmids as polymerization templates for mutagenesis. This method can directly create user-defined mutation libraries on the plasmid. It offers broad sequence programmability and even covers methylated plasmids. We further combine this method with rational design to engineer genome-editing protein FnCpf1. The FnCpf1 variants with mutations in the PAM-binding groove were generated. Several variants expanded the PAM range and exhibited lower off-target effect, which can loosen PAM constraint and enhance target specificity for genome editing. This work presents an effective tool for plasmid mutagenesis and protein engineering studies.

Microneedle Array-Assisted, Direct Delivery of Genome-Editing Proteins Into Plant Tissue

Genome editing in plants employing recombinant DNA often results in the incorporation of foreign DNA into the host genome. The direct delivery of genome-editing proteins into plant tissues is desired to prevent undesirable genetic alterations. However, in most currently available methods, the point of entry of the genome-editing proteins cannot be controlled and time-consuming processes are required to select the successfully transferred samples. To overcome these limitations, we considered a novel microneedle array (MNA)-based delivery system, in which the needles are horizontally aligned from the substrate surface, giving it a comb-like configuration. We aimed to deliver genome-editing proteins directly into the inner layers of leaf tissues; palisade, the spongy and subepidermal L2 layers of the shoot apical meristem (SAM) which include cells that can differentiate into germlines. The array with needles 2 μm wide and 60 μm long was effective in inserting into Arabidopsis thaliana leaves and Glycine max (L.) Merr. (soybeans) SAM without the needles buckling or breaking. The setup was initially tested for the delivery of Cre recombinase into the leaves of the reporter plant A. thaliana by quantifying the GUS (β-glucuronidase) expression that occurred by the recombination of the loxP sites. We observed GUS expression at every insertion. Additionally, direct delivery of Cas9 ribonucleoprotein (RNP) targeting the PDS11/18 gene in soybean SAM showed an 11 bp deletion in the Cas9 RNP target site. Therefore, this method effectively delivered genome-editing proteins into plant tissues with precise control over the point of entry.

TALEN outperforms Cas9 in editing heterochromatin target sites

Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.

Protein Delivery: Intracellular Delivery of His‐Tagged Genome‐Editing Proteins Enabled by Nitrilotriacetic Acid–Containing Lipidoid Nanoparticles (Adv. Healthcare Mater. 6/2019)

In article number 1800996, Qiaobing Xu and co-workers report a new type of non-cationic lipidoid nanoparticle for His-tagged protein delivery. Active lipidoids are synthesized by conjugating nitrilotriacetic acid with hydrophobic tails and nanoparticles are formulated in the presence of helper lipids through self-assembly. The newly developed lipidoid nanoparticles are capable of delivering genome-editing proteins (Cre recombinase and CRISPR/Cas9 ribonucleoprotein) into mammalian cells.

Nanoscale ATP-Responsive Zeolitic Imidazole Framework-90 as a General Platform for Cytosolic Protein Delivery and Genome Editing.

Metal-organic frameworks (MOFs) are an emerging class of nanocarriers for drug delivery, owing to their tunable chemical functionality. Here we report ATP-responsive zeolitic imidazole framework-90 (ZIF-90) as a general platform for cytosolic protein delivery and CRISPR/Cas9 genome editing. The self-assembly of imidazole-2-carboxaldehyde and Zn2+ with protein forms ZIF-90/protein nanoparticles and efficiently encapsulates protein. It was found that, in the presence of ATP, the ZIF-90/protein nanoparticles are degraded to release protein due to the competitive coordination between ATP and the Zn2+ of ZIF-90. Intracellular delivery studies showed that the ZIF-90/protein nanoparticle can deliver a large variety of proteins into the cytosol, regardless of protein size and molecular weight. The delivery of cytotoxic RNase A efficiently prohibits tumor cell growth, while the effective delivery of genome-editing protein Cas9 knocks out the green fluorescent protein (GFP) expression of HeLa cells with efficiency up to 35%. Given the fact that ATP is upregulated in disease cells, it is envisaged that the ATP-responsive protein delivery will open up new opportunities for an advanced protein delivery and CRISPR/Cas9 genome editing for targeted disease treatment.

Integrating Combinatorial Lipid Nanoparticle and Chemically Modified Protein for Intracellular Delivery and Genome Editing.

The use of protein to precisely manipulate cell signaling is an effective approach for controlling cell fate and developing precision medicine. More recently, programmable nucleases, such as CRISPR/Cas9, have shown extremely high potency for editing genetic flow of mammalian cells, and for treating genetic disorders. The therapeutic potential of proteins with an intracellular target, however, is mostly challenged by their low cell impermeability. Therefore, a developing delivery system to transport protein to the site of action in a spatiotemporal controlled manner is of great importance to expand the therapeutic index of the protein. In this Account, we first summarize our most recent advances in designing combinatorial lipid nanoparticles with diverse chemical structures for intracellular protein delivery. By designing parallel Michael addition or ring-opening reaction of aliphatic amines, we have generated a combinatorial library of cationic lipids, and identified several leading nanoparticle formulations for intracellular protein delivery both in vitro and in vivo. Moreover, we optimized the chemical structure of lipids to control lipid degradation and protein release inside cells for CRISPR/Cas9 genome-editing protein delivery. In the second part of this Account, we survey our recent endeavor in developing a chemical approach to modify protein, in particular, coupled with the nanoparticle delivery platform, to improve protein delivery for targeted diseases treatment and genome editing. Chemical modification of protein is a useful tool to modulate protein function and to improve the therapeutic index of protein drugs. Herein, we mostly summarize our recent advances on designing chemical approaches to modify protein with following unique findings: (1) chemically modified protein shows selective turn-on activity based on the specific intracellular microenvironment, with which we were able to protein-based targeted cancer therapy; (2) the conjugation of hyaluronic acid (HA) to protein allows cancer cell surface receptor-targeted delivery of protein; (3) the introduction of nonpeptidic boronic acid into protein enabled cell nucleus targeted delivery; this is the first report that a nonpeptidic signal can direct protein to subcellular compartment; and (4) the fusion of protein with negatively supercharged green fluorescent protein (GFP) facilitates the self-assembly of protein with lipid nanoparticle for genome-editing protein delivery. At the end of the Account, we give a perspective of expanding the chemistry that could be integrated to design biocompatible lipid nanocarriers for protein delivery and genome editing in vitro and in vivo, as well as the chemical approaches that we can harness to modulate protein activity in live cells for targeted diseases treatment.

Intracellular Delivery of His-Tagged Genome-Editing Proteins Enabled by Nitrilotriacetic Acid-Containing Lipidoid Nanoparticles.

Protein- and peptide-based therapeutics with high tolerance and specificity along with low off-target effects and genetic risks have attracted tremendous attention over the last three decades. Herein, a new type of noncationic lipidoid nanoparticle (LNP) is reported for His-tagged protein delivery. Active lipidoids are synthesized by conjugating a nitrilotriacetic acid group with hydrophobic tails (EC16, O16B, and O17O) and nanoparticles are formulated in the presence of nickel ions and helper lipids (cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). It is demonstrated that the newly developed LNPs are capable of delivering various His-tagged proteins including green fluorescent protein (GFP), (-30)GFP-Cre recombinase, and CRISPR/Cas9 ribonucleoprotein into mammalian cells.

Introduction of pathogenic mutations into the mouse Psen1 gene by Base Editor and Target-AID

Base Editor (BE) and Target-AID (activation-induced cytidine deaminase) are engineered genome-editing proteins composed of Cas9 and cytidine deaminases. These base-editing tools convert C:G base pairs to T:A at target sites. Here, we inject either BE or Target-AID mRNA together with identical single-guide RNAs (sgRNAs) into mouse zygotes, and compare the base-editing efficiencies of the two distinct tools in vivo. BE consistently show higher base-editing efficiency (10.0–62.8%) compared to that of Target-AID (3.4–29.8%). However, unexpected base substitutions and insertion/deletion formations are also more frequently observed in BE-injected mice or zygotes. We are able to generate multiple mouse lines harboring point mutations in the mouse presenilin 1 (Psen1) gene by injection of BE or Target-AID. These results demonstrate that BE and Target-AID are highly useful tools to generate mice harboring pathogenic point mutations and to analyze the functional consequences of the mutations in vivo.

Combinatorial library of chalcogen-containing lipidoids for intracellular delivery of genome-editing proteins

Protein based therapeutics with high specificities and low off-target effects are used for transient and accurate manipulation of cell functions. However, developing safe and efficient carriers for intracellular delivery of active therapeutic proteins is a long-standing challenge. Here we report a combinatorial library of chalcogen (O, S, Se) containing lipidoid nanoparticles (LNPs) as efficient nanocarriers for intracellular delivery of negatively supercharged Cre recombinase ((-30)GFP-Cre) and anionic Cas9:single-guide RNA (Cas9:sgRNA) ribonucleoprotein (RNP) for genome editing. The structure-activity relationship between the lipidoids and intracellular protein delivery efficiencies was explored and it was demonstrated that the newly developed LNPs are effective for gene recombination in vivo.

Manufacturing and Delivering Genome-Editing Proteins.

Genome-editing technologies have revolutionized the biomedical sciences by providing researchers with the ability to quickly and efficiently modify genes. While programmable nucleases can be introduced into cells using a variety of techniques, their delivery as purified proteins is an effective approach for limiting off-target effects. Here, we describe step-by-step procedures for manufacturing and delivering genome-modifying proteins-including Cas9 ribonucleoproteins (RNPs) and TALE and zinc-finger nucleases-into mammalian cells. Protocols for combining Cas9 RNP with naturally recombinogenic adeno-associated virus (AAV) donor vectors for the seamless insertion of transgenes by homology-directed genome editing are also provided.

Small molecule-triggered Cas9 protein with improved genome-editing specificity.

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen (4-HT)-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.