Cas9 Endonuclease Research Articles

Reactive oxygen signaling molecule inducible regulation of CRISPR-Cas9 gene editing.

We report development of a controllable gene editing tool that boronated gRNA, simply generated in situ, could regulate binding of gRNA molecules with either Cas9 endonuclease or target genes, thus serving as a modulator that can control CRISPR-Cas9 gene editing. Subsequent treatment with H2O2 facilitates the restoration of gene editing ability of the boronated gRNA to the level of using untreated gRNA. This is one of the few cases using small molecule to regulate CRISPR-Cas9 gene editing, which is a complement to the light approach, displaying great application potential. We develop a controllable gene editing tools based on the CRISPR-Cas9 gene editing system. This tool can be regulated by oxidative small molecule, i.e., H2O2. Compared with the light method, the application scope of our CRISPR-Cas9 systems have been widened with the small-molecule-triggered approaches, preventing the potential damage of cells or organism caused by UV light. In addition, the gain-of-function tools are expanding the gene code expansion for mechanistic studies of target enzymes since it provides a positive route to evaluate the activity of a given enzyme in dynamic and inversible regulation of targeting cellular processes.

Modelling the Applications of CRISPR‐Cas9 for Treating Sickle Cell Disease

Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR‐associated protein 9 (CRISPR‐Cas9) is a genome editing tool that can remove, modify, or add sections of DNA. It is made up of two main molecules: the Cas9 protein and the guide‐RNA (gRNA), which is used to match a target gene to the CRISPR‐Cas9 molecule. The desired sequence of DNA must be between 2 and 5 nucleotides and must be followed by the protospacer adjacent motif (PAM) at the 3’ end of the gRNA. This enables the target gene to be found and cut by the Cas9 endonuclease. Once cut, the DNA can be repaired using non‐homologous end joining (where a random insertion or deletion of DNA occurs) or homology directed repair (where a homologous piece of DNA is used as a template to repair the DNA). The latter is used for genome editing, as the target sequence can be changed with the CRISPR‐Cas9 system.Sickle Cell Disease (SCD) is an autosomal recessive hereditary disorder that affects erythrocytes and is caused by a single base substitution of adenine to thymine on the hemoglobin beta (HbB) gene. Currently two different groups are examining two different techniques to treat SCD. One group has shown that by deleting the BCL11A gene, fetal hemoglobin production can occur, which greatly diminishes the amount of Sickle hemoglobin (HbS). An example of this application of CRISPR‐Cas9 for treating SCD is the story of Victoria Gray, the first person to have received genome editing treatment for SCD.

A Quantitative Comparison of the Knockdown Efficiencies of CRISPR/Cas9 and CRISPR‐Cas12 in Caenorhabditis elegans

Currently, many gene editing studies employ CRISPR/Cas9, however its low accuracy and knockdown efficiency is problematic. CRISPR‐Cas12, a newer and potentially more accurate variant, is hypothesized to have a higher knockdown efficiency than CRISPR/Cas9 in Saccharomyces cerevisiae and various plants. However this has not been quantitatively verified in mammalian cells. Our study compares the loss of gene function efficiencies of CRISPR/Cas9 and CRISPR‐Cas12 through decreasing green fluorescent protein (GFP) expression in Caenorhabditis elegans via CRISPR‐mediated deletion. We used the LP162 strain of C. elegans which endogenously expresses GFP. We used C. elegans due to the similarity of molecular pathways to those of humans, their simple anatomy, and their fast reproductive cycle. Through bacterial transformation, we expressed our modified plasmid (containing the gRNA sequence, Cas endonuclease sequence, and C. elegans‐specific promoters) in Escherichia coli. The C. elegans subsequently ingested the transformed bacteria. Fluorescence and protein expression were used as assays to determine the efficacy of CRISPR‐mediated GFP deletion. We hope to show a quantitative assessment of the difference in loss of function efficiencies between CRISPR/Cas9 and CRISPR‐Cas12 when used to delete GFP in C. elegans. Our goal is for these findings to be valuable for researchers seeking to use the most efficient Cas endonuclease for gene knockdown experiments.

A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells

The CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing. An important drawback of the CRISPR-Cas9 system is the constitutive endonuclease activity when Cas9 endonuclease and its sgRNA are co-expressed. This constitutive activity results in undesirable off-target effects that hinder studies using the system, such as probing gene functions or its therapeutic use in humans. Here, we describe a convenient method that allows temporal and tight control of CRISPR-Cas9 activity by combining transcriptional regulation of Cas9 expression and protein stability control of Cas9 in human stem cells. To achieve this dual control, we combined the doxycycline-inducible system for transcriptional regulation and FKBP12-derived destabilizing domain fused to Cas9 for protein stability regulation. We showed that approximately 5%–10% of Cas9 expression was observed when only one of the two controls was applied. By combining two systems, we markedly lowered the baseline Cas9 expression and limited the exposure time of Cas9 endonuclease in the cell, resulting in little or no undesirable on- or off-target effects. We anticipate that this dual conditional CRISPR-Cas9 system can serve as a valuable tool for systematic characterization and identification of genes for various pathological processes.

Cas9-mediated maternal effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae.

CRISPR/Cas9 technologies are important tools for the development of gene-drive systems to modify mosquito vector populations to control the transmission of pathogens that cause diseases such as malaria. However, one of the challenges for current Cas9-based drive systems is their ability to produce drive-resistant alleles resulting from insertions and deletions (indels) caused principally by nonhomologous end-joining following chromosome cleavage. Rapid increases in the frequency of such alleles may impair gene-drive dynamics. We explored the generation of indels in the germline and somatic cells in female gene-drive lineages using a series of selective crosses between a gene-drive line, AgNosCd-1, and wild-type mosquitoes. We find that potential drive-resistant mutant alleles are generated largely during embryonic development, most likely caused by deposition of the Cas9 endonuclease and guide RNAs in oocytes and resulting embryos by homozygous and hemizygous gene-drive mothers.

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Cas13d: A New Molecular Scissor for Transcriptome Engineering.

The discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated Cas endonucleases in bacterial and archaeal species allowed scientists to modify, utilized, and revolutionize this tool for genetic alterations in any species. Especially the type II CRISPR-Cas9 system has been extensively studied and utilized for precise and efficient DNA manipulation in plant and mammalian systems over the past few decades. Further, the discovery of the type V CRISPR-Cas12 (Cpf1) system provides more flexibility and precision in DNA manipulation in prokaryotes, plants, and animals. However, much effort has been made to employ and utilize the above CRISPR tools for RNA manipulation but the ability of Cas9 and Cas12 to cut DNA involves the nuisance of off-target effects on genes and thus may not be employed in all RNA-targeting applications. Therefore, the search for new and diverse Cas effectors which can precisely detect and manipulate the targeted RNA begins and this led to the discovery of a novel RNA targeting class 2, type VI CRISPR-Cas13 system. The CRISPR-Cas13 system consists of single RNA-guided Cas13 effector nucleases that solely target single-stranded RNA (ssRNA) in a programmable way without altering the DNA. The Cas13 effectors family comprises four subtypes (a-d) and each subtype has distinctive primary sequence divergence except the two consensuses Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) that includes RNase motifs i.e. R-X4-6-H. These two HEPN domains are solely responsible for executing targetable RNA cleavage activity with high efficiency. Further, recent studies have shown that Cas13d exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. In addition to that, Cas13d has shown additional advantages over other Cas13 variants, structurally as well as functionally which makes it a prominent and superlative tool for RNA engineering and editing. Therefore considering the advantages of Cas13d over previously characterized Cas13 subtypes, in this review, we encompass the structural and mechanistic properties of type VI CRISPR-Cas13d systems, an overview of the current reported various applications of Cas13d, and the prospects to improve Cas13d based tools for diagnostic and therapeutic purposes.

A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and can characterize any CRISPR-Cas nuclease – benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range

The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

Convergent Arrangement of sgRNA and Cas9 in CRISPRsome for Transcellular Trafficking

The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing systems has led to the appreciation of CRISPR as a versatile and powerful gene-editing tool in the molecular biology laboratory and a potentially potent therapeutic for the treatment of a variety of intractable diseases. Nonetheless, one of the most onerous obstacles that restricts the wide availability of CRISPR technology from wide practical applications is the lack of appropriate delivery vehicles for the simultaneous transportation of multicomponent CRISPR systems to the intracellular targets. Herein, we attempted to create an intriguing nanometer-scaled vesicle platform (CRISPRsome) that, with the aid of a block copolymer of poly(ethylene glycol)-polylysine, simultaneously combined the fundamental components of the CRISPR system into the CRISPRsome, particularly the ribonucleic sgRNA and proteinic Cas9 endonuclease precisely located at the intermediate membrane and the internal reservoir regions of the nanometer-scaled vesicles, respectively. Furthermore, biofunctionalization strategies, including using redox-responsive disulfide linkages between lysine units to cross-link the well-defined CRISPRsome structures and surface functionalization with the cyclic Arg-Gly-Asp ligand to stimulate receptor-mediated endocytosis, were proposed in the well-defined CRISPRsome. The subsequent results validated its appreciable gene knockout efficacies toward cancerous cells with the green fluorescent protein (GFP) reporter gene, thereby highlighting the prosperous potential of CRISPRsome to facilitate the use of CRISPR technology in a variety of practical applications.

Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?

Simple SummaryThe advent of genomic editing with CRISPR/Cas9 has transformed the way we manipulate the genome, and has facilitated the investigation of tumor cell biology in vitro and in vivo. Not only we can modify genome sequence to blunt an overactivated gene or correct a mutation, but also we may modulate gene expression using CRISPR/Cas system. In this review, we present the basics of CRISPR/Cas methodology, its components and how to start a CRISPR/Cas experiment; Moreover, we present how CRISPR/Cas methodology has been applied to study the function of coding and noncoding genes in thyroid cancer and provided insights into cancer biology.Important advances on the role of genetic alterations in thyroid cancer have been achieved in the last two decades. One key reason is linked to the development of technical approaches that allowed for the mimicking of genetic alterations in vitro and in vivo and, more recently, the gene editing methodology. The CRISPR/Cas methodology has emerged as a tangible tool for editing virtually any DNA sequence in the genome. To induce a double-strand break and programmable gene editing, Cas9 endonuclease is guided by a single-guide RNA (sgRNA) that is complementary to the target sequence in DNA. The gene editing per se occurs as the cells repair the broken DNA and may erroneously change the original DNA sequence. In this review, we explore the principles of the CRISPR/Cas system to facilitate an understanding of the mainstream technique and its applications in gene editing. Furthermore, we explored new applications of CRISPR/Cas for gene modulation without changing the DNA sequence and provided a Dry Lab experience for those who are interested in starting “CRISPRing” any given gene. In the last section, we will discuss the progress in the knowledge of thyroid cancer biology fostered by the CRISPR/Cas gene editing tools.

Pinpoint modification strategy for stabilization of single guide RNA

The clustered regularly interspaced short palindromic repeats-CRISPR associated protein9 (CRISPR-Cas9) system, which includes a single guide RNA (sgRNA) and a Cas9 protein, is an emerging and promising gene editing technology that produces specific changes, including insertions, deletions, or substitutions, in desired targets. This approach can be applied in novel therapeutic areas for multiple cancers and genetic diseases, including Parkinson’s disease, sickle cell disease, and muscular dystrophy. However, there are many limitations to its potential application to therapeutics. CRISPR-Cas9 activity without side effects, delivery of CRISPR-Cas9 to the target cell within the desired tissue including liver, lungs, brain and muscle and the expression of Cas9 endonuclease in the target cell are key factors in achieving therapeutic efficacy. Generally, single-stranded RNA is immediately degraded in cells and biological fluids such as serum, as chemically unmodified single-stranded RNA shows extremely poor stability against nuclease degradation. To overcome this limitation, sgRNA is chemically modified to obtain a highly stable sgRNA for efficient gene editing in cells and in vivo. Here, we identified the cleavage site of sgRNA for pinpoint modification in biological tissues using mass spectrometry and improved stability of pinpoint modified sgRNA in these fluids. Although improved efficiency provided by modified sgRNA has already been reported, we identified the cleavage site by mass spectrometry and revealed that the stability increased with the pinpoint modification strategy for the first time in this study. In future studies, the efficiency of pinpoint modification strategy for the potential application of sgRNA by systematic routes, including intravenous and subcutaneous administration will be assessed.

An update on precision genome editing by homology-directed repair in plants.

Beneficial alleles derived from local landraces or related species, or even orthologs from other plant species, are often caused by differences of one or several single-nucleotide polymorphisms or indels in either the promoter region or the encoding region of a gene and often account for major differences in agriculturally important traits. Clustered regularly interspaced short palindromic repeats-associated endonuclease Cas9 system (CRISPR/Cas9)-mediated precision genome editing enables targeted allele replacement or insertion of flag or foreign genes at specific loci via homology-directed repair (HDR); however, HDR efficiency is low due to the intrinsic rare occurrence of HDR and insufficient DNA repair template in the proximity of a double-stranded break (DSB). Precise replacement of the targeted gene with elite alleles from landraces or relatives into a commercial variety through genome editing has been a holy grail in the crop genome editing field. In this update, we briefly summarize CRISPR/Cas-mediated HDR in plants. We describe diverse strategies to improve HDR efficiency by manipulating the DNA repair pathway, timing DSB induction, and donor delivery, and so on. Lastly, we outline open questions and challenges in HDR-mediated precision genome editing in both plant biological research and crop improvement.

Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.

Distribution, Diversity and Roles of CRISPR-Cas Systems in Human and Animal Pathogenic Streptococci.

Streptococci form a wide group of bacteria and are involved in both human and animal pathologies. Among pathogenic isolates, differences have been highlighted especially concerning their adaptation and virulence profiles. CRISPR-Cas systems have been identified in bacteria and many streptococci harbor one or more systems, particularly subtypes I-C, II-A, and III-A. Since the demonstration that CRISPR-Cas act as an adaptive immune system in Streptococcus thermophilus, a lactic bacteria, the diversity and role of CRISPR-Cas were extended to many germs and functions were enlarged. Among those, the genome editing tool based on the properties of Cas endonucleases is used worldwide, and the recent attribution of the Nobel Prize illustrates the importance of this tool in the scientific world. Another application is CRISPR loci analysis, which allows to easily characterize isolates in order to understand the interactions of bacteria with their environment and visualize species evolution. In this review, we focused on the distribution, diversity and roles of CRISPR-Cas systems in the main pathogenic streptococci.

Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes

SummaryObjectiveMicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes.MethodPrimary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140−/− mice.ResultsHigh efficiency gene editing of miR-140 (90–98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium.ConclusionWe describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.

Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen-Deuterium Exchange Mass Spectrometry.

The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.

Applications of CRISPR/Cas9 technology for modification of the plant genome.

The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) system was discovered in bacteria and archea as an acquired immune response to protect the cells from infection. This technology has now evolved to become an efficient genome editing tool, and is replacing older gene editing technologies. This technique uses programmable sgRNAs to guide the Cas9 endonuclease to the target DNA location. sgRNA is a vital component of the CRISPR technology, since without it the Cas nuclease cannot reach to its target location. Over the years, many tools have been developed for designing sgRNAs, the details of which have been extensively reviewed here. It has proven to be a promising tool in the field of genetic engineering and has successfully generated many plant varieties with better and desirable qualities. In the present review, we attempted to collect,collate and summarize information related to the development of CRISPR/Cas9 system as a tool and subsequently into a technique having a wide array of applications in the field of plant genome editing in attaining desirable traits like resistance to various diseases, nutritional enhancement etc. In addition, the probable future prospects and the various bio-safety concerns associated with CRISPR gene editing technology have been discussed in detail.

CRISPR/Cas9-Mediated Genome Editing System in Insect Genomics and Pest Management.

The CRISPR/Cas9 is being developed as an invaluable system that allows rapid and site-specific genome editing in a wide variety of organisms, including diverse insects. It has been successfully used for gene function annotations of RNAi pathway in insect genomics and will facilitate research on RNAi mechanism. Here, we describe a streamlined method to generate and detect somatic and germline knockout mutations of desired target genes in tephritid pests by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed single guide RNA (sgRNA) into embryos. Target site selection, sgRNA synthesis, Cas9 synthesis, microinjection, and mutation identification are presented in detail.

DNA-functionalized colloidal crystals for macromolecular encapsulation.

Novel DNA-based structures with the ability to encapsulate nanoscale molecules, such as proteins, can be applied to a wide range of areas, including reaction fields and micro/nano drug carriers. DNA-functionalized nanoparticle (DNA-NP) colloidal crystals have emerged as a new class of programmable DNA-based structures harboring metal nanoparticles with improved mechanical properties. The encapsulation of guest molecules into empty spaces in lattice structures is theoretically possible. However, due to the lack of a strategy for versatile encapsulation of guest molecules, the feasibility of nanoscale encapsulation by DNA-NP crystals is unclear. In this study, we developed DNA-functionalized gold nanoparticle (DNA-AuNP) crystals with tunable interparticle spacing for molecular encapsulation. We demonstrated that the modification of DNA-AuNP crystals with functional moieties, that is, biotin molecules, was effective in retaining molecules in the crystals. The crystallinities before and after encapsulation of the molecules were confirmed using small-angle X-ray scattering. We also succeeded in encapsulating CRISPR/Cas9 ribonucleoproteins into DNA-AuNP crystals by harnessing their affinity for target molecules. These findings demonstrated the potential use of metal-DNA hybrid crystals as carriers for direct protein delivery via biolistic bombardment. Thus, this study provides an attractive strategy for creating a new class of DNA-based structures for macromolecular encapsulation, and an alternative research direction toward colloidal crystal engineering using DNA.