Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.

Genome editing technologies that install diverse edits can widely enable genetic studies and new therapeutics. Here we develop click editing, a genome writing platform that couples the advantageous properties of DNA-dependent DNA polymerases with RNA-programmable nickases (e.g. CRISPR-Cas) to permit the installation of a range of edits including substitutions, insertions, and deletions. Click editors (CEs) leverage the "click"-like bioconjugation ability of HUH endonucleases (HUHes) with single stranded DNA substrates to covalently tether "click DNA" (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs (DNA polymerase and HUHe orthologs, architectural modifications, etc.) and their clkDNAs (template configurations, repair evading substitutions, etc.), we demonstrate the ability to install precise genome edits with minimal indels and no unwanted byproduct insertions. Since clkDNAs can be ordered as simple DNA oligonucleotides for cents per base, it is possible to screen many different clkDNA parameters rapidly and inexpensively to maximize edit efficiency. Together, click editing is a precise and highly versatile platform for modifying genomes with a simple workflow and broad utility across diverse biological applications.

Precise DNA cleavage using CRISPR-SpRYgests.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

Effects of treatment methods on the formation of resistant starch in purple sweet potato.

In order to determine the mechanisms underlying resistant starch formation, three treatments were used to prepare resistant starch from purple sweet potato. The resistant starch yield, amylose content, chain length distribution, thermal properties, and crystal structure were determined, and the results were compared with those of unmodified starch. Autoclaving, pullulanase, and pullulanase-autoclaving treatments significantly increased the resistant starch yield, amylose content, shorter amylopectin branch content, and gelatinisation temperatures of native purple sweet potato starch. Resistant starch prepared via pullulanase-autoclaving combination treatment exhibited the highest gelatinisation enthalpy value and the greatest degree of overall thermal stability. X-ray diffraction patterns and Fourier-transform infrared spectra analysis demonstrated that all three treatments transformed the starch crystalline structure from C-type to B-type, and no new groups were generated during the modification process; all the processes were only physical modifications.

Single-Atom Zinc and Anionic Framework as Janus Separator Coatings for Efficient Inhibition of Lithium Dendrites and Shuttle Effect.

The two key problems for the industrialization of Li-S batteries are the dendrite growth of lithium anode and the shuttle effect of lithium polysulfides (LiPSs). Herein, we report the Janus separator prepared by coating anionic Bio-MOF-100 and its derived single-atom zinc catalyst on each side of the Celgard separator. The anionic metal-organic framework (MOF) coating induces the uniform and rapid deposition of lithium ions, while its derived single-atom zinc catalyzes the rapid transformation of LiPSs, thus inhibiting the lithium dendrite and shuttle effect simultaneously. Consequently, compared with other reported Li-S batteries assembled with single-atomic catalysts as separator coatings, our SAZ-AF Janus separator showed stable cyclic performance (0.05% capacity decay rate at 2 C with 1000 cycles), outstanding performance in protecting lithium anode (steady cycle 2800 h at 10 mAh cm-2), and equally excellent cycling performance in Li-SeS2 or Li-Se batteries. Our work provides an effective separator coating design to inhibit shuttle effect and lithium dendrite.

Development of the Nude Rabbit Model.

Loss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.

MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9's homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a "plug and play" module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

CRISPR/Cas9-Mediated TERT Disruption in Cancer Cells.

Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Efficient Gene Editing at Major CFTR Mutation Loci.

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163Mut/Mut pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163Mut/Mut pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163Mut/Mut pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.

Screen and Verification for Transgene Integration Sites in Pigs.

Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs. After CRISPR/Cas9-mediated site-specific integration on Pifs501, we detected high EGFP expression in different pig cell types and ubiquitous EGFP expression in diverse tissues of transgenic pigs without adversely affecting 600 kb neighboring gene expression. Promoters integrated on Pifs501 exhibit hypomethylated modification, which suggest a permissive epigenetic status of this locus. We establish a versatile master cell line on Pifs501, which allows us to achieve site-specific exchange of EGFP to Follistatin with Cre/loxP system conveniently. Through in vitro and in vivo functional assays, we demonstrate the effectiveness of this screening method, and take Pifs501 as a potential site for transgene insertion in pigs. We anticipate that Pifs501 will have useful applications in pig genome engineering, though the identification of genomic safe harbor should over long-term various functional studies.