Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple “unlinked” transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called “inteins” can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a “chaining” design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.
Albert Cheng, PhD 郑瑚博士
Professor
Institute of Zoology, CAS
研究员
中国科学院动物研究所
Synthetic Biology + Genome Engineering
Publication Types:
Synthetic BZLF1-targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers
The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.
Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold
RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3’ end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.
Multiplex RNA targeting
Provided herein, in some aspects, is a multiplex RNA targeting system that enables live cell imaging and/or modification of multiple RNA targets. Specifically, the disclosure provides a method of live cell imaging of ribonucleic acid (RNA), or targeting RNA in a live cell, comprising: (a) delivering to a cell an RNA-editing complex that comprises a catalytically inactive Cas13 (dCas13) nuclease, a Cas 13 guide RNA (gRNA) comprising an RNA aptamer sequence, and a detectable molecule linked to an RNA-binding domain (RBD), or an RNA effector molecule linked to an RBD sequence that specifically binds to the RNA aptamer sequence; and (b) imaging the detectable molecule or RNA aptamer and RBD binding.
Targeted sequence insertion compositions and methods
Targeted directional kilobase sequence insertion by combining prime editing with recombinases or integrases
Targeted insertion of exogenous sequences to genomes is useful for therapeutics and biological research. While CRISPR/Cas technologies have been very efficient in gene knockouts by double-strand breaks (DSBs) followed by indel formation through non-homologous end-joining (NHEJ) repair pathway, the precise introduction of new sequences mainly rely on inefficient homology directed repair (HDR) pathways following Cas9-induced DSBs and are restricted to dividing cells. The recent invention of Prime Editing allows short sequences to be precisely inserted at target sites without DSBs. Here, we combine Prime Editing and sequence-specific recombinases and integrases to insert kilobase sequences directionally at target sites. This technique, called PRIMAS for Prime editing, Recombinase, Integrase-mediated Addition of Sequence, will expand our genome editing toolbox for targeted insertion of long sequences up to kilobases and beyond.
Local recruitment of DNA repair proteins enhances CRISPR-ssODN-HDR editing
CRISPR-Cas technologies enable precise editing of genomic sequences. One major way to introduce precise editing is through homology directed repair (HDR) of DNA double strand breaks (DSB) templated by exogenously supplied single-stranded oligodeoxyribonucleotides (ssODN). Competing pathways determine the outcome of edits. Non-homologous end-joining pathways produce destructive insertions/deletions (indels) at target sites and are dominant over the precise homology directed repair pathways. In this study, we aim to favor HDR and use two strategies to recruit DNA repair proteins (DRPs) to Cas9 cut site, the Casilio-DRP approach that recruits RNA-binding protein-tethered DRPs to target site via aptamers appended to guide RNA; and the 53BP1-DRP approach that recruits DRPs to DSBs via DSB-sensing activity of 53BP1. We conducted two screens using these approaches and identified DRPs such as FANCF and BRCA1 that when recruited to Cas9 cut site, enhance ssODN-templated HDR and increase the proportion of precise edits. This study provides not only new constructs for enhanced CRISPR-ssODN-HDR but also a collection of DRP fusions for studying DNA repair processes.
JACKIE: Fast Enumeration of Genome-Wide Single- and Multicopy CRISPR Target Sites and Their Off-Target Numbers
Zinc finger protein-, transcription activator like effector-, and CRISPR-based methods for genome and epigenome editing and imaging have provided powerful tools to investigate functions of genomes. Targeting sequence design is vital to the success of these experiments. Although existing design software mainly focus on designing target sequence for specific elements, we report here the implementation of Jackie and Albert’s Comprehensive K-mer Instances Enumerator (JACKIE), a suite of software for enumerating all single- and multicopy sites in the genome that can be incorporated for genome-scale designs as well as loaded onto genome browsers alongside other tracks for convenient web-based graphic-user-interface-enabled design. We also implement fast algorithms to identify sequence neighborhoods or off-target counts of targeting sequences so that designs with low probability of off-target can be identified among millions of design sequences in reasonable time. We demonstrate the application of JACKIE-designed CRISPR site clusters for genome imaging.
CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus
Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.
TALE.Sense: A Versatile DNA Sensor Platform for Live Mammalian Cells
Methods and Compositions for Recruiting DNA Repair Proteins
The DNA repair complexes provided herein are, inter alia, useful for editing genome sequences by introducing precise changes in a target site in the presence of a donor sequence. The RNA-guided DNA endonuclease provided herein including embodiments thereof (e.g., Cas9 nuclease or Cas9 nickase) is capable of introducing a strand break (double- or single-strand break) at a target site in the genome of a cell (e.g., gene or transcriptional regulatory sequence) and the break is then predominantly repaired through the mechanism of HDR. By increasing the HDR efficiency significantly and in certain instances decreasing NHEJ at a target site, the compositions and methods provided herein meet the long-felt need of site directed, highly accurate genome editing.
Artificial RNA-guided splicing factors
Provided herein, in some aspects, are compositions and methods for artificially modulating alternative splicing, for example, inducing exon inclusion and/or exon exclusion events. In some embodiments, a catalytically inactive programmable nuclease, such as dCasRx, is fused to an RNA-binding protein (or fragment or isoform thereof) and, when guided to a target of interest by a specific guide RNA (gRNA), can regulate alternative splicing in eukaryotic cells.
Transgenic selection methods and compositions
The present disclosure provides a split intein selectable marker system for the production and selection of transgenic cells.
Split Selectable Markers
Sequence detection systems
The present disclosure, in some embodiments, provides sequence detection systems (sequence detectors) for the detection of specific nucleotides sequences present in the genome of live cells (e.g., single live cells) to achieve, for example, in vivo and in situ imaging, cell selection, and/or cell ablation.
Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways
Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.
Targeted Enhanced DNA Demethylation
Provided herein are, inter alia, compositions and methods for the delivery of enhanced demethylation activity to target DNA sequences in a mammalian cell. The compositions and methods are, useful for activity modulation of a targeted gene, or to create a gene regulatory network.
Targeted DNA demethylation and methylation
Provided herein are, inter alia, compositions and methods for demethylating and methylating a target DNA sequences in a mammalian cell. The compositions and methods are, useful for activity modulation of a targeted gene, or to create a gene regulatory network.
CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells
CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells
Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling
Abstract
The RNA-guided DNA endonuclease system CRISPR-Cas9 has been exploited for
genome editing in various species. The nuclease-deficient mutant dCas9 protein can,
when coupled with sgRNAs, bind specific genomic loci without inducing DNA cleavage,
thus serving as a programmable DNA binding protein. To extend the utility of the dCas9
system, we have taken advantage of the ability of Pumilio PUF domains to bind specific
8-mer RNA sequences. By combining these two systems, we established the Casilio
system, which allows for specific and independent delivery of effector proteins to
specific genomic loci. We demonstrated that the Casilio system enables independent upand
down-regulation of multiple genes, as well as live-cell imaging of multiple genomic
loci simultaneously. Importantly, multiple copy of PUF binding sites can be incorporated
on sgRNA backbone, therefore allowing for local multimerization of effectors. In
addition, the PUF domain can be engineered to recognize any 8-mer RNA sequence,
therefore enabling the generation and simultaneous operation of many Casilio modules.
A website specifically for Casilio is at http://casil.io
A three-component CRISPR/Cas complex system and uses thereof
The invention described herein provides compositions and reagents for assembling a tripartite complex at a specific location of a target DNA. The invention also provides methods for using the complex to, for example, label a specific genomic locus, to regulate the expression of a target gene, or to create a gene regulatory network.
Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease
CRISPR/Cas is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 mRNA, sgRNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted NHEJ and HDR mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.
Methods of mutating, modifying or modulating nucleic acid in a cell or nonhuman mammal
The invention is directed to a method of mutating one or more target nucleic acid sequences in a stem cell or a zygote comprising introducing into the stem cell or zygote (i) ribonucleic acid (RNA) sequences that comprise a portion that is complementary to a portion of each of the target nucleic acid sequences and comprise a binding site for a CRISPR associated (Cas) protein; and a Cas nucleic acid sequence or a variant thereof that encodes a Cas protein having nuclease activity. The stem cell or zygote is maintained under conditions in which the target nucleic acid sequences are mutated in the stem cell or zygote. The invention is also directed to methods of producing a non human mammal carrying mutations and methods of modulating the expression and/or activity target nucleic acid sequences and cells or zygotes.
CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice
OBJECTIVE:
To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing.
APPROACH AND RESULTS:
The CRISPR/Cas9 system was used to produce 3 of 18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell-restricted Cnn1 gene. Each founder was bred for germline transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1(ΔCArG/ΔCArG)) mice. Quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1(ΔCArG/ΔCArG) mice with no change in other smooth muscle cell-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of serum response factor to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall.
CONCLUSIONS:
CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in smooth muscle cell DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease.
TALEN-mediated editing of the mouse Y chromosome
The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes–Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.
Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system
Abstract
Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.