Cas9 Ribonucleoprotein Research Articles

Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish.

Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.

NanoRNP Overcomes Tumor Heterogeneity in Cancer Treatment.

Tumor heterogeneity has been one of the most important factors leading to the failure of conventional cancer therapies due to the accumulation of genetically distinct tumor-cell subpopulations during the tumor development process. Due to the diversity of genetic mutations during tumor growth, combining the use of multiple drugs has only achieved limited success in combating heterogeneous tumors. Herein, we report a novel antitumor strategy that effectively addresses tumor heterogeneity by using a CRISPR/Cas9-based nanoRNP carrying a combination of sgRNAs. Such nanoRNP is synthesized from Cas9 ribonucleoprotein, any combinations of required sgRNAs, and a rationally designed responsive polymer that endows nanoRNP with high circulating stability, enhanced tumor accumulation, and the efficient gene editing in targeted tumor cells eventually. By carrying a combination of sgRNAs that targets STAT3 and RUNX1, the nanoRNP exhibited efficient gene expression disruptions on a heterogeneous tumor model with two subsets of cells whose proliferations were sensitive to the reduced expression of STAT3 and RUNX1, respectively, leading to the effective growth inhibition of the heterogeneous tumor. Considering the close relationship between tumor heterogeneity and cancer progression, resistance to therapy, and recurrences, nanoRNP provides a feasible strategy to overcome tumor heterogeneity in the development of more advanced cancer therapy against malignant tumors.

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.

DNA-free mutagenesis of GIGANTEA in Brassica oleracea var. capitata using CRISPR/Cas9 ribonucleoprotein complexes

The CRISPR–Cas9 system is a powerful tool for editing genes of interest in specific plant genomes. Indeed, genome-editing systems have been used to enhance a variety of agricultural traits and to study gene functions in many plant species, and the plasmid-mediated delivery of Cas9 and single guide RNA (sgRNA) to plants has been reported to facilitate highly efficient editing. However, the random and stable integration of plasmid DNA sequences into plant genomes can cause insertional mutagenesis, and an additional step is required to remove such foreign sequences from edited plant genomes. Accordingly, the aim of the present study was to investigate the effectiveness of directly delivering purified CRISPR–Cas9 ribonucleoproteins (RNPs) to protoplasts from cabbage (Brassica oleracea var. capitata), an important cruciferous vegetable. The flowering-time regulator gene GIGANTEA (GI) was targeted, with the goal of delaying flowering time and prolonging vegetative growth. We investigated the targeted mutagenesis insertion and deletion rates using targeted deep sequencing. The mutation frequency achieved using one of the sgRNAs (sgRNA2) was 2% in the infected protoplast. The shoots were regenerated from 44% (46/103) of protoplast-derived calli. Consequently, three independent and completely transgene-free mutants were obtained, including one homogeneous biallelic line in which both GI alleles were successfully edited, thereby yielding a complete GI knockout line. These results suggest that the transgene-free CRISPR–Cas9 system is a promising tool for improving agricultural beneficial traits of cabbage.

Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9.

Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host’s tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the C. hominivorax and L. cuprina yellow genes (ChY and LcY) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation (brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the C. hominivorax transformer gene (Chtra). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.

Delivering Cas9/sgRNA ribonucleoprotein (RNP) by lentiviral capsid-based bionanoparticles for efficient 'hit-and-run' genome editing.

Transient expression of the CRISPR/Cas9 machinery will not only reduce risks of mutagenesis from off-target activities, but also decrease possible immune response to Cas9 protein. Building on our recent developing of a system able to package up to 100 copies of Cas9 mRNA in each lentivirus-like particle (LVLP) via the specific interaction between aptamer and aptamer-binding proteins (ABP), here we develop a lentiviral capsid-based bionanoparticle system, which allows efficient packaging of Cas9/sgRNA ribonucleoprotein (RNP). We show that replacing the Tetraloop of sgRNA scaffold with a com aptamer preserves the functions of the guide RNA, and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like particles via the specific interactions between ABP and aptamer, and sgRNA and Cas9 protein. These RNP bionanoparticles generated Indels on different targets in different cells with efficiencies similar to or better than our recently described Cas9 mRNA LVLPs. The new system showed fast action and reduced off-target rates, and makes it more convenient and efficient in delivering Cas9 RNPs for transient Cas9 expression and efficient genome editing.

A carrier-free multiplexed gene editing system applicable for suspension cells

Genetically engineered cells via CRISPR/Cas9 system can serve as powerful sources for cancer immunotherapeutic applications. Furthermore, multiple genetic alterations are necessary to overcome tumor-induced immune-suppressive mechanisms. However, one of the major obstacles is the technical difficulty with efficient multiple gene manipulation of suspension cells due to the low transfection efficacy. Herein, we established a carrier-free multiplexed gene editing platform in a simplified method, which can enhance the function of cytotoxic CD8+ T cells by modulating suspension cancer cells. Our multiple Cas9 ribonucleoproteins (RNPs) enable simultaneous disruption of two programmed cell death 1 (PD-1) ligands, functioning as negative regulators in the immune system, by accessing engineered Cas9 proteins with abilities of complexation and cellular penetration. In addition, combination with electroporation enhanced multiple gene editing efficacy, compared with that by treatment of multiple Cas9 RNPs alone. This procedure resulted in high gene editing at multiple loci of suspension cells. The treatment of multiple Cas9 RNPs targeting both ligands strongly improved Th1-type cytokine production of cytotoxic CD8+ T cells, resulting in synergistic cytotoxic effects against cancer. Simultaneous suppression of PD-L1 and PD-L2 on cancer cells via our developed editing system allows effective anti-tumor immunity. Furthermore, the treatment of multiple Cas9 RNPs targeting PD-L1, PD-L2, and TIM-3 had approximately 70–90% deletion efficacy. Thus, our multiplexed gene editing strategy endows potential clinical utilities in cancer immunotherapy.

Cas9 Ribonucleoprotein Complex Delivery: Methods and Applications for Neuroinflammation.

The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract.

S852 IN VIVO SELECTION FOR CORRECTED BETA‐GLOBIN ALLELES AFTER CRISPR/CAS9 EDITING IN HUMAN SICKLE HEMATOPOIETIC STEM CELLS ENHANCES THERAPEUTIC POTENTIAL

Background:Sickle Cell Disease (SCD) is a devastating disorder caused by a single base change in the ß‐globin gene (HBB) that is one component of the adult hemoglobin tetramer (HbA, which contains 2 a‐globin and 2 ß‐globin proteins). SCD is a recessive disorder – if one of the 2 alleles of HBB is normal, the disease is not present; thus gene correction needs to correct only one of the two HBB alleles in any given stem cell.Aims:To correct the sickle allele in human hematopoietic stem cellsMethods:To correct the sickle mutation, we developed a process that directly introduces a preassembled Cas9 ribonucleoprotein (RNP) complex (composed of recombinant Cas9 protein and single guide RNA) along with a single‐stranded DNA oligonucleotide donor template (ssODN) for homology‐directed repair (HDR). We studied gene correction after long‐term xenografting of human sickle HSCs. After electroporation with RNP and ssODN, sickle human HSCs were injected into immunodeficient NBSGW mice, which permit engraftment of human HSCs and erythroid differentiation in the bone marrow.Results:Sixteen to 20 weeks after injection, we analyzed engrafted human cells in the bone marrow of 43 mice in 4 cohorts. We quantified editing in xenografted cells, and found an average of 23.4% of HBB alleles with the corrected genotype, and 65.2% with insertion/deletion mutations (indels). To assess the distribution of HBB alleles within populations of xenografted cells, we inferred HBB genotypes using RNA‐Seq data from 359 individual clonal BFU‐E colonies derived from marrow CD34+ cells (figure 1A). This reveals that a substantial proportion of corrected (HDR) alleles are heterozygous with indel alleles equivalent to ß‐thalassemia mutations, confirming that corrected (wild type) HBB alleles in edited HSCs are distributed such that many erythrocytes have only a single wild type HBB allele. Data from the colony analysis, extrapolated to the entire experimental group, indicates that the 23.4% of corrected HBB alleles are distributed among >30% of HSCs, producing a comparable proportion of functional erythrocytes. SCD is a recessive disorder; based upon observations of mixed chimerism after allogeneic transplantation, this proportion of normal cells is consistent with cure.From engrafted NBSGW mice, we immunoselected human erythroid cells with anti‐CD235a (GlycophorinA), carried out RNA‐Seq, and inferred HBB genotypes in the erythroblast population. For each individual mouse, we compared the proportion of corrected HBB alleles in the xenografted bone marrow, in CD34+ cells immunoselected from marrow, and in CD235a+ erythroblasts. To control for enrichment of alleles corrected by HDR, we distinguished two types of HDR: “PAM‐only” HDR events mutate the Cas9 PAM without the conversion tract reaching the sickle mutation, whereas “sickle‐corrected” HDR events mutate the Cas9 PAM and also correct the sickle mutation. We observe a marked enrichment of “sickle‐corrected” HDR alleles in erythroblasts when compared to marrow or CD34+ cells from the same mouse, but no enrichment of “PAM‐only” HDR alleles (figure 1B).Summary/Conclusion:In summary, we have developed a Cas9‐mediated gene editing protocol that reproducibly yields a cell product in which the sickle mutation is corrected to wild type at a level that is sufficient to cure SCD. Evidence we have presented indicates that cells carrying the corrected ß‐globin gene are enriched during in vivo erythroid differentiation. These results support moving this therapeutic approach to the clinic.image

Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.

Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia

AbstractThe thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.

High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins

Efficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to efficiently induce homologous recombination. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins

CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

SummaryWe describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes

BackgroundThe CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.ResultsIn this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated.ConclusionsThe developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

Targeted Gene Disruption in Pacific Oyster Based on CRISPR/Cas9 Ribonucleoprotein Complexes.

The Pacific oyster (Crassostrea gigas) is a representative bivalve mollusc that is widely cultured in the world. In recent years, it has become an important model species for ecological, evolutionary, and developmental studies because of its ability to survive in extreme environmental conditions as a sessile filter feeder and its classical mosaic pattern of development. Although the complete genome sequence of C. gigas is available and omics data have been rapidly generated for the past few years, the genetic tools for gene functional studies have thus far been limited to RNA interference technology. In this study, we developed a gene editing system for C. gigas based on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 ribonucleoprotein complexes. Two candidate genes, myostatin (MSTN) and Twist, were selected as targets. After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, CRISPR-induced indel mutations were detected in the target genes. The CRISPR/Cas9-induced mutations were predominantly small indel mutations ranging in size from 1 to 24bp in these two target genes. These results demonstrate that CRISPR/Cas9 can be successfully used as an effective targeted gene editing system in C. gigas. The method reported here provides a powerful tool for gene functional studies in oysters and other marine bivalves, and potentially as a new technology for genetic engineering to improve oyster traits for aquaculture.

Efficient Intracellular Delivery of CRISPR-Cas Ribonucleoproteins through Receptor Mediated Endocytosis.

We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.

CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice.

Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos' genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation embryos with zona pellucida, glycoprotein membrane surrounding early embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donor DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.