Exonuclease editor promotes precision of gene editing in mammalian cells.

BACKGROUND: Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. RESULTS: Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. CONCLUSIONS: Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

Enhancing prime editor flexibility with coiled-coil heterodimers.

BACKGROUND: Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency. RESULTS: Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol. CONCLUSIONS: Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos.

Cas9 protein without sgRNAs can induce genomic damage at the cellular level in vitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9 protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

VCFshiny: an R/Shiny application for interactively analyzing and visualizing genetic variants.

Summary: Next-generation sequencing generates variants that are typically documented in variant call format (VCF) files. However, comprehensively examining variant information from VCF files can pose a significant challenge for researchers lacking bioinformatics and programming expertise. To address this issue, we introduce VCFshiny, an R package that features a user-friendly web interface enabling interactive annotation, interpretation, and visualization of variant information stored in VCF files. VCFshiny offers two annotation methods, Annovar and VariantAnnotation, to add annotations such as genes or functional impact. Annotated VCF files are deemed acceptable inputs for the purpose of summarizing and visualizing variant information. This includes the total number of variants, overlaps across sample replicates, base alterations of single nucleotides, length distributions of insertions and deletions (indels), high-frequency mutated genes, variant distribution in the genome and of genome features, variants in cancer driver genes, and cancer mutational signatures. VCFshiny serves to enhance the intelligibility of VCF files by offering an interactive web interface for analysis and visualization. Availability and implementation: The source code is available under an MIT open source license at https://github.com/123xiaochen/VCFshiny with documentation at https://123xiaochen.github.io/VCFshiny.

BCL11B and the NuRD complex cooperatively guard T-cell fate and inhibit OPA1-mediated mitochondrial fusion in T cells.

The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T-cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)-cell fate in T cells. In addition, T cells upregulate the NK cell-associated receptors and transcription factors, lyse NK-cell targets, and are reprogrammed into NK-like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1-mediated elevated OXPHOS enhances cellular acetyl-CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T-cell fate directly by repressing NK cell-associated transcription and indirectly through a metabolic-epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs.

Precise genome editing of the Kozak sequence enables bidirectional and quantitative modulation of protein translation to anticipated levels without affecting transcription.

None of the existing approaches for regulating gene expression can bidirectionally and quantitatively fine-tune gene expression to desired levels. Here, on the basis of precise manipulations of the Kozak sequence, which has a remarkable influence on translation initiation, we proposed and validated a novel strategy to directly modify the upstream nucleotides of the translation initiation codon of a given gene to flexibly alter the gene translation level by using base editors and prime editors. When the three nucleotides upstream of the translation initiation codon (named KZ3, part of the Kozak sequence), which exhibits the most significant base preference of the Kozak sequence, were selected as the editing region to alter the translation levels of proteins, we confirmed that each of the 64 KZ3 variants had a different translation efficiency, but all had similar transcription levels. Using the ranked KZ3 variants with different translation efficiencies as predictors, base editor- and prime editor-mediated mutations of KZ3 in the local genome could bidirectionally and quantitatively fine-tune gene translation to the anticipated levels without affecting transcription in vitro and in vivo. Notably, this strategy can be extended to the whole Kozak sequence and applied to all protein-coding genes in all eukaryotes.

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation.

BACKGROUND: CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. RESULTS: Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. CONCLUSIONS: Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.

Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.

Generation of a genetically modified pig model with CREBRFR457Q variant.

Obesity is among the strongest risk factors for type 2 diabetes (T2D). The CREBRF missense allele rs373863828 (p. Arg457Gln, p. R457Q) is associated with increased body mass index but reduced risk of T2D in people of Pacific ancestry. To investigate the functional consequences of the CREBRF variant, we introduced the corresponding human mutation R457Q into the porcine genome. The CREBRFR457Q pigs displayed dramatically increased fat deposition, which was mainly distributed in subcutaneous adipose tissue other than visceral adipose tissue. The CREBRFR457Q variant promoted preadipocyte differentiation. The increased differentiation capacity of precursor adipocytes conferred pigs the unique histological phenotype that adipocytes had a smaller size but a greater number in subcutaneous adipose tissue (SAT) of CREBRFR457Q variant pigs. In addition, in SAT of CREBRFR457Q pigs, the contents of the peroxidative metabolites 4-hydroxy-nonenal and malondialdehyde were significantly decreased, while the activity of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase, was increased, which was in accordance with the declined level of the reactive oxygen species (ROS) in CREBRFR457Q pigs. Together, these data supported a causal role of the CREBRFR457Q variant in the pathogenesis of obesity, partly via adipocyte hyperplasia, and further suggested that reduced oxidative stress in adipose tissue may mediate the relative metabolic protection afforded by this variant despite the related obesity.

AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns.

Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression.

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Eliminating predictable DNA off-target effects of cytosine base editor by using dual guiders including sgRNA and TALE.

Predictable DNA off-target effect is one of the major safety concerns for the application of cytosine base editors (CBEs). To eliminate Cas9-dependent DNA off-target effects, we designed a novel effective CBE system with dual guiders by combining CRISPR with transcription activator-like effector (TALE). In this system, Cas9 nickase (nCas9) and cytosine deaminase are guided to the same target site to conduct base editing by single-guide RNA (sgRNA) and TALE, respectively. However, if nCas9 is guided to a wrong site by sgRNA, it will not generate base editing due to the absence of deaminase. Similarly, when deaminase is guided to a wrong site by TALE, base editing will not occur due to the absence of single-stranded DNA. In this way, Cas9- and TALE-dependent DNA off-target effects could be completely eliminated. Furthermore, by fusing TALE with YE1, a cytidine deaminase with minimal Cas9-independent off-target effect, we established a novel CBE that could induce efficient C-to-T conversion without detectable Cas9- or TALE-dependent DNA off-target mutations.

Human induced-T-to-natural killer cells have potent anti-tumour activities.

BACKGROUND: Adoptive cell therapy (ACT) is a particularly promising area of cancer immunotherapy, engineered T and NK cells that express chimeric antigen receptors (CAR) are being explored for treating hematopoietic malignancies but exhibit limited clinical benefits for solid tumour patients, successful cellular immunotherapy of solid tumors demands new strategies. METHODS: Inactivation of BCL11B were performed by CRISPR/Cas9 in human T cells. Immunophenotypic and transcriptional profiles of sgBCL11B T cells were characterized by cytometer and transcriptomics, respectively. sgBCL11B T cells are further engineered with chimeric antigen receptor. Anti-tumor activity of ITNK or CAR-ITNK cells were evaluated in preclinical and clinical studies. RESULTS: We report that inactivation of BCL11B in human CD8+ and CD4+ T cells induced their reprogramming into induced T-to-natural killer cells (ITNKs). ITNKs contained a diverse TCR repertoire; downregulated T cell-associated genes such as TCF7 and LEF1; and expressed high levels of NK cell lineage-associated genes. ITNKs and chimeric antigen receptor (CAR)-transduced ITNKs selectively lysed a variety of cancer cells in culture and suppressed the growth of solid tumors in xenograft models. In a preliminary clinical study, autologous administration of ITNKs in patients with advanced solid tumors was well tolerated, and tumor stabilization was seen in six out nine patients, with one partial remission. CONCLUSIONS: The novel ITNKs thus may be a promising novel cell source for cancer immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03882840 . Registered 20 March 2019-Retrospectively registered.

In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers.

BACKGROUND: Mutations in the DMD gene encoding dystrophin-a critical structural element in muscle cells-cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. METHODS: In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46-54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. RESULTS: Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member ß-dystroglycan. CONCLUSIONS: We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

CRISPR/Cas9-Mediated Gene Correction in Newborn Rabbits with Hereditary Tyrosinemia Type I.

Patients with hereditary tyrosinemia type I (HT1) present acute and irreversible liver and kidney damage during infancy. CRISPR-Cas9-mediated gene correction during infancy may provide a promising approach to treat patients with HT1. However, all previous studies were performed on adult HT1 rodent models, which cannot authentically recapitulate some symptoms of human patients. The efficacy and safety should be verified in large animals to translate precise gene therapy to clinical practice. Here, we delivered CRISPR-Cas9 and donor templates via adeno-associated virus to newborn HT1 rabbits. The lethal phenotypes could be rescued, and notably, these HT1 rabbits reached adulthood normally without 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione administration and even gave birth to offspring. Adeno-associated virus (AAV)-treated HT1 rabbits displayed normal liver and kidney structures and functions. Homology-directed repair-mediated precise gene corrections and non-homologous end joining-mediated out-of-frame to in-frame corrections in the livers were observed with efficiencies of 0.90%-3.71% and 2.39%-6.35%, respectively, which appeared to be sufficient to recover liver function and decrease liver and kidney damage. This study provides useful large-animal preclinical data for rescuing hepatocyte-related monogenetic metabolic disorders with precise gene therapy.

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems.

BACKGROUND: Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. RESULTS: In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. CONCLUSION: The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation.

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells. A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs. Therefore, to achieve a transplantable organ in animals without rejection, creation of vascular endothelial cells derived from humans within the organ is necessary. In this study, to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals, we generated rat-mouse chimeras by injection of rat embryonic stem cells (rESCs) into mouse blastocysts with deficiency of Flk-1 protein, which is associated with endothelial and hematopoietic cell development. We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras. The whole yolk sac (YS) of Flk-1EGFP/EGFP rat-mouse chimera was full of rat blood vasculature. Rat genes related to vascular endothelial cells, arteries, and veins, blood vessels formation process, as well as hematopoietic cells, were highly expressed in the YS. Our results suggested that rat vascular endothelial cells could undergo proliferation, migration, and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells, T cells, and myeloid cells in rat-mouse chimeras, which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.