Lentiviral-based vectors, related systems, and methods for gene editing in eukaryotes

Abstract

Providing lentiviral-based vectors, related systems, and methods for gene editing in eukaryotes. [Solution] A composition, system, and method useful for achieving gene editing in eukaryotic cells are provided. The composition comprises a plasmid encoding one or more viral fusion proteins in which one or more viral proteins are fused with an aptamer-binding protein. The composition also comprises a plasmid encoding a nonviral nucleic acid sequence in which a nonviral nucleic acid sequence encodes a component of the CRISPR system. In some cases, the nonviral nucleic acid sequence also comprises an aptamer sequence. The plasmid can be used to produce viral particles containing lentivirus-like particles containing viral fusion proteins and nonviral RNA sequences. A system for producing such viral particles is provided.

Description

[Technical Field]

[0001] Prior related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 665,080, filed on 1 May 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002] The clustered, regularly interspaced short palindromic repeats (CRISPR) / CRISPR-related (Cas) system, discovered in bacteria, can be used as a tool for modifying mammalian and human genomes for gene therapy, gene expression regulation, and DNA and RNA labeling. In the CRISPR / Cas system, CRISPR-related nucleases are targeted to genomic sites by forming a complex with guide RNA (gRNA) that hybridizes with the target site in the genome. This results in a double-strand break, initiating either non-homologous end joining (NHEJ) or homology-directed repair (HDR) of genomic DNA via a double-stranded or single-stranded DNA template. Modified systems using nickase to introduce single-strand breaks (modified CRISPR-related nucleases) have also been developed.

[0003] Currently, the standard practice is to deliver DNA expressing CRISPR-related nucleases via plasmid DNA, lentivirus, or adeno-associated virus. All of these delivery systems are plagued by the risk of mutagenesis due to off-target and prolonged nuclease expression. Therefore, efficient transient delivery of CRISPR / Cas9 components is necessary. [Overview of the project] [Means for solving the problem]

[0004] This disclosure relates to compositions, systems, and methods useful for achieving gene editing in eukaryotic cells. In some cases, the compositions, systems, and methods may be used to package CRISPR-related endonuclease mRNA into viral particles, such as lentiviral particles. In some cases, the compositions, systems, and methods may be used to package CRISPR-related endonuclease and gRNA sequences into viral particles, for example, as a ribonucleoprotein complex. The compositions include plasmids encoding one or more viral fusion proteins in which one or more viral proteins are fused with an aptamer-binding protein. The compositions also include plasmids encoding nonviral nucleic acid sequences in which nonviral nucleic acid sequences encode components of the CRISPR system. In some cases, the nonviral nucleic acid sequences also include aptamer sequences. In some cases, the nucleic acid encoding the CRISPR-related endonuclease includes at least one aptamer sequence. In some examples, the gRNA coding sequence includes at least one aptamer sequence. The plasmids may be used to produce viral particles containing lentiviral-like particles. Systems for producing such viral particles are provided. Furthermore, methods for using the viral particles described herein to achieve gene editing in eukaryotic cells are also provided.

[0005] Furthermore, a lentiviral packaging plasmid is provided that includes a eukaryotic cell promoter operably linked to a Gag nucleotide sequence, the Gag nucleotide sequence comprising a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, where one or both of the NC coding sequence or MA coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence, and the packaging plasmid does not encode a functional integrase protein.

[0006] Further provided are mammalian expression plasmids comprising a eukaryotic cell promoter operably linked to a viral protein R (VPR) coding sequence or a negative regulator (NEF) coding sequence, wherein the VPR coding sequence or NEF coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence.

[0007] Also provided are mammalian expression plasmids comprising a eukaryotic cell promoter operably ligated to a nonviral nucleic acid sequence, the nonviral nucleic acid sequence comprising at least one aptamer coding sequence, the nonviral nucleic acid sequence comprising (i) one or both of a CRISPR-related endonuclease coding sequence or a guide RNA (gRNA) coding sequence and (ii) at least one aptamer coding sequence.

[0008] A lentiviral packaging system is further provided, comprising: a) a packaging plasmid comprising a eukaryotic cell promoter operably ligated to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, and one or both of the NC coding sequence or the MA coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence, and the packaging plasmid does not encode a functional integrase protein; b) a mammalian expression plasmid comprising a eukaryotic cell promoter operably ligated to a nonviral nucleic acid sequence, wherein the nonviral nucleic acid sequence comprises a CRISPR-related endonuclease coding sequence, a guide RNA (gRNA) coding sequence, or both a CRISPR-related endonuclease coding sequence and a gRNA coding sequence; and c) an envelope plasmid comprising an envelope glycoprotein coding sequence.

[0009] Also provided is a lentiviral packaging system comprising: a) a packaging plasmid that does not encode a functional integrase protein; b) a mammalian expression plasmid comprising a eukaryotic cell promoter operably linked to a viral protein R (VPR) coding sequence or a negative regulator (NEF) coding sequence, wherein one or both of the VPR coding sequence or the NEF coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence; c) a mammalian expression plasmid comprising a eukaryotic cell promoter operably linked to a nonviral nucleic acid sequence, wherein the nonviral nucleic acid sequence comprises a CRISPR-related endonuclease coding sequence, a guide RNA (gRNA) coding sequence, or both a CRISPR-related endonuclease coding sequence and a gRNA coding sequence; and d) an envelope plasmid comprising an envelope glycoprotein coding sequence.

[0010] Furthermore, the invention also provides lentivirus-like particles comprising a) a fusion protein comprising a nucleocapsid (NC) protein or a matrix (MA) protein, wherein the NC protein or MA protein comprises at least one nonviral aptamer-binding protein (ABP); and b) at least one nonviral RNA molecule wherein the nonviral RNA sequence comprises CRISPR-related endonuclease mRNA, guide RNA (gRNA), or both CRISPR-related endonuclease mRNA and gRNA, and which do not contain a functional integrase protein.

[0011] The invention also provides a lentivirus-like particle comprising a) a fusion protein containing a viral protein R (VPR) protein or a negative regulator (NEF) protein, wherein the VPR protein or NEF protein contains at least one nonviral aptamer-binding protein (ABP), and b) at least one nonviral RNA molecule, wherein the nonviral RNA sequence contains CRISPR-related endonuclease mRNA, guide RNA (gRNA), or both CRISPR-related endonuclease mRNA and gRNA, and which does not contain a functional integrase protein.

[0012] The invention also provides lentivirus-like particles comprising a) a fusion protein containing a viral protein R (VPR) protein or a negative regulator (NEF) protein, wherein the VPR protein or NEF protein contains at least one nonviral aptamer-binding protein (ABP), and b) a ribonucleotide protein (RNP) complex containing a CRISPR-related endonuclease and guide RNA, but without a functional integrase protein.

[0013] Also provided is a method for producing lentiviral particles, comprising: a) transfecting a plurality of eukaryotic cells with a packaging plasmid, at least one mammalian expression plasmid, and an envelope plasmid from any one of the systems described herein; and b) culturing the transfected eukaryotic cells for a time sufficient to produce lentiviral particles.

[0014] Furthermore, a method for producing lentiviral particles is also provided, comprising: a) transfecting a plurality of eukaryotic cells with a plasmid from any of the systems described herein; and b) culturing the transfected eukaryotic cells for a time sufficient to produce lentiviral particles.

[0015] Furthermore, a method for modifying a genome target sequence in cells, comprising transduction of multiple eukaryotic cells using multiple viral particles, wherein the multiple viral particles include i) a lentivirus-like particle described herein, wherein the nonviral RNA sequence comprises CRISPR-related endonuclease mRNA and gRNA, or ii) a lentivirus-like particle described herein, wherein the nonviral RNA sequence comprises CRISPR-related endonuclease mRNA and the second viral particle comprises gRNA or a gRNA coding sequence, and CRISPR-related endonuclease However, a method is also provided in which, in cells transduced using lentivirus-like particles, CRISPR-related endonuclease mRNA is expressed, and if the second viral particle contains a gRNA coding sequence, the gRNA is expressed from the gRNA coding sequence in cells transduced using the second viral particle, and the CRISPR-related endonuclease and gRNA form a complex that binds to a genomic target sequence in the cell's genomic DNA, and the CRISPR-related endonuclease cleaves the cell's genomic DNA, thereby inducing a cellular DNA repair mechanism and causing modification of the genomic target sequence.

[0016] Furthermore, a method for modifying a genomic target sequence in a cell is provided, comprising transducing a plurality of eukaryotic cells using a plurality of viral particles, wherein the plurality of viral particles include lentivirus-like particles containing a ribonucleotide protein (RNP) complex (a CRISPR-related endonuclease complexed with at least one gRNA), the RNP binding to the genomic target sequence in the cell's genomic DNA, and the CRISPR-related endonuclease cleaving the cell's genomic DNA, thereby inducing a cellular DNA repair mechanism and causing modification of the genomic target sequence. Cells modified by any of the genomic modifications described herein are also provided. Cells containing any of the lentivirus-like particles described herein are also provided.

[0017] Furthermore, there are also methods for treating a disease in a subject, comprising: a) obtaining cells from the subject; b) modifying the cells of the subject using one of the methods for modifying genomic target sequences described herein; and c) administering the modified cells to the subject. In some cases, the disease is cancer. In some cases, the cells are T cells.

[0018] This disclosure includes the following figures. The figures are intended to illustrate the characteristics of certain embodiments and / or compositions and methods, and to supplement any description of the compositions and methods. The figures do not limit the scope of the compositions and methods unless the description expressly indicates otherwise. [Brief explanation of the drawing]

[0019] [Figure 1-1]Figures 1A and 1B are schematic diagrams illustrating compositions, systems, and methods according to aspects of the present disclosure. As shown in Figure 1A, a plasmid is used to express a lentiviral protein fused with an aptamer-binding protein (ABP), referred to in the figure as a viral protein ABP fusion protein. The plasmid encoding the viral protein ABP fusion protein coding sequence may be a lentiviral packaging plasmid (e.g., for NC or MA fusion proteins) or a mammalian expression vector (e.g., for NEF or VPR fusion proteins). Figure 1A also shows a mammalian expression plasmid containing nonviral nucleic acid sequences encoding CRISPR system components (i.e., CRISPR-related endonucleases and sgRNA) that are used together with the packaging plasmid and envelope plasmid to transfect eukaryotic cells and generate lentiviral-like particles containing the viral protein ABP fusion protein and nonviral RNA molecules (i.e., CRISPR-related endonuclease mRNA, sgRNA, or both). Optionally, the nonviral RNA molecules encoded by the mammalian expression plasmid may include aptamer sequences. If the viral protein ABP fusion protein coding sequence is provided in a mammalian expression plasmid (e.g., for NEF and VPR fusion proteins), a lentiviral packaging plasmid is also used to transfect eukaryotic cells. The mammalian expression plasmid may be a lentiviral transfer plasmid encoding various viral genes. The packaging plasmids (whether or not they contain the viral protein ABP fusion protein coding sequence) and the transfer plasmid (if used) may be modified to remove the viral sequence to produce lentiviral-like particles. The lentiviral-like particles are recovered from eukaryotic cells. If necessary, sgRNA may be packaged in the lentiviral particles, or the sgRNA coding sequence may be packaged in a DNA virus instead of lentiviral-like particles.

[0020] [Figure 1-2] Figures 1A and 1B are schematic diagrams illustrating compositions, systems, and methods according to embodiments of the present disclosure. As shown in Figure 1B, lentivirus-like particles may be used to transduce eukaryotic cells, thereby providing CRISPR-related endonuclease mRNA and sgRNA molecules from which an active protein is translated in the cells. These nonviral RNA molecules may be provided in the same lentivirus-like particle or in different viral particles. The CRISPR-related endonuclease protein and sgRNA interact to form a ribonucleoprotein complex that binds to a genomic target site (as directed by the sgRNA) and introduces a cleavage in the DNA. The cleavage can be repaired by homologous recombination repair (HDR) or non-homologous end joining (NHEJ) mechanisms. If HDR is desired, eukaryotic cells may be transduced using viral particles containing a target template sequence that can be used as a template for modification of the genomic target site. Since the amount of CRISPR-related endonuclease mRNA introduced into the cells is finite, it is eventually degraded as the protein is expressed from the mRNA. If necessary, sgRNA may be provided as a synthetic RNA molecule introduced into cells via transfection, either within lentiviral particles or DNA viral particles, or, if cells are cultured. Figure 1B shows in vitro or ex vivo transduction of eukaryotic cells, while the described viral particles may be administered to subjects to induce in vivo gene editing.

[0021] [Figure 2] Figure 2 is a schematic diagram of an MS2 coat protein (MCP) fusion protein for SaCas9 mRNA packaging in lentivirus-like particles according to certain aspects of this disclosure. VPR: viral protein R, NEF: negative regulator; NC: nucleocapsid protein; MA: matrix protein. Dashed lines indicate deleted regions.

[0022] [Figure 3]Figure 3 is a graph illustrating how the addition of an HBB3'UTR sequence to SaCas9 mRNA improved the gene editing activity of virus-like particles according to certain aspects of this disclosure. Different amounts of enriched virus-like particles containing SaCas9 mRNA with two copies of the HBB 3'UTR sequence following the MS2 aptamer sequence were simultaneously transduced into 2.5 × 10⁴ GFP reporter cells using an equal amount of lentiviral-like vector expressing HBB sgRNA1. GFP-positive cells were detected by flow cytometry. Each data point was the average of two replicates.

[0023] [Figure 4] Figure 4 is an image illustrating the production of lentivirus-like particles from MCP- and PCP-based lentivirus packaging plasmids according to aspects of this disclosure. The control lentivirus particles had a lentivirus genome expressing GFP. The MCP- and PCP-based lentivirus-like particles contained SaCas91xMS2 mRNA and SaCas91xPP7 mRNA, respectively. The mean ± sem of the control, MCP-, and PCP-based particles were 98.5 ± 10.5, 105.7 ± 3.1, and 123.9 ± 5.8 nm, respectively, n ≥ 11.

[0024] [Figure 5] Figures 5A and 5B are graphs illustrating transient expression of SaCas9 mRNA in HEK293T cells transduced using different viral vectors according to aspects of this disclosure. Figure 5A shows the expression of SaCas9 mRNA from adeno-associated virus (AAV) and embedded-defective lentivirus (IDLV) vectors. Figure 5B shows the expression of SaCas9 mRNA from lentivirus-like particles containing SaCas91xPP7, SaCas91xMS2, or SaCas91xPP7-3'UTR. RNA levels were measured by quantitative RT-PCR 24, 48, 72, and 96 hours after transduction. * indicates p<0.05 between SaCas91xMS2 and SaCas91xMS2-2×3'UTR by bidirectional ANOVA.

[0025] [Figure 6] Figure 6 shows indels observed in the HBB sgRNA1 target sequence of GFP reporter cells transduced using lentivirus-like particles according to an aspect of this disclosure. GFP reporter cells were co-transduced using lentivirus-like particles containing SaCas91xMS2-3'UTR mRNA and a lentivirus expressing HBB-sgRNA1. DNA was amplified by PCR and sequenced by next-generation sequencing. The top 10 most observed sequences are listed as SEQ ID NOs. 103-112 (from top to bottom). 13.5% of alleles were unmodified, and all others contained indels. The target sequence is underlined, and the PAM sequence is the 6 nucleotides preceding the target sequence. Arrows indicate predicted cleavage sites. The wild-type HBB sequence has an "A" at the second position of the target sequence; a "T" at that position indicates a nucleotide difference compared to the wild-type HBB sequence. Data were derived from 5,774,277 sequence read data.

[0026] [Figure 7-1]Figures 7A–7F are schematic diagrams illustrating the compositions, systems, and results obtained using LVLP for SaCas9 mRNA packaging. Figure 7A shows the MCP-viral protein fusion protein and SaCas9-MS2 aptamer fusion RNA (SaCas9 fused to SEQ ID NO: 211) for packaging SaCas9 mRNA into LVLP. MCP: MS2 coat protein; VPR: viral protein R, NEF: negative regulator; NC: nucleocapsid protein; MA: matrix protein. The dashed line indicates a deleted region in MA. Figure 7B shows the plasmid for producing lentivirus and LVLP. The aptamer-binding protein MCP and MS2 aptamer are shown. LTR: terminal repeat sequence; Ψ: lentiviral packaging signal. Figure 7C shows the effect of the MCP fusion protein on lentivirus production. 5 × 10⁵ cells in a 6-well plate were transfected with the plasmid shown (total DNA 5 μg). 24 hours after transfection, the medium was replaced with 2 ml of fresh medium, and p24 was assayed after 24 hours. Individual data points and mean ± SEM are shown. Only the MA-MCP modification reduced virus production (p<0.05). Figure 7D shows that the best gene editing activity was obtained when MCP was fused to NC. SaCas91xMS2 was expressed during LVLP production. 250 μl of LVLP-containing supernatant and 250 μl of IDLV-expressing HBB sgRNA1 were simultaneously transduced into 2.5 × 10⁴ GFP reporter cells. ** indicates p<0.01 when LVLP from NC-MCP fusion is compared to other particles; * indicates p<0.05 when compared to any other particle. Figure 7E shows flow cytometry analysis of GFP reporter cells transduced with or without NC-MCP-modified packaging plasmids. The supernatant, with increased volume, was used to simultaneously transduce GFP reporter cells with 50 ng of HBB sgRNA1-expressing IDLV.When particles were produced without using a packaging plasmid, the packaging plasmid was replaced with pKanCMV-mRuby3-10aa-H2B expressing mRuby. ***, p<0.001, when comparing the GFP positivity rates of the two conditions (Bonferroni post-test following ANOVA). Figure 7F shows that the plasmid DNA contributed little to the generation of GFP-positive reporter cells. pSaCas91xMS2 (column 1) or pSaCas91xMS2-HBB sgRNA1 plasmid DNA (column 2) were transfected into GFP reporter cells to observe GFP-positive cells. pSaCas91xMS2-HBB sgRNA1 was also used to construct LVLPs for transduction into GFP reporter cells using an IDLV expressing HBB sgRNA1 alone (100ng p24, column 3) or 50ng p24 (column 4). *** indicates p<0.001. For Figures 7C, 7D, and 7F, Tukey's multiple comparison test was performed following the ANOVA analysis. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0027] [Figure 8] Figure 8 shows that when GFP reporter cells were transduced using the supernatant generated by replacing the packaging plasmid with an mRuby expression plasmid (located in the nucleus), very few mRuby-positive cells were observed. Shown is an image from one field of view of cells treated with 534 μl of supernatant. Multiple fields of view were examined, and 0–5 positive cells were observed per field of view. Three fields of view, each containing 2, 2, and 3 positive cells, were estimated by imageJ to contain 934 cells / field of view. The mean positivity rate was 0.0025%.

[0028] [Figure 9]Figures 9A-9E show the gene editing activity of various LVLPs. Figure 9A shows the effect of MS2 aptamers on SaCas9 mRNA levels. Plasmid DNA expressing SaCas9nxMS2 (250 ng) was co-transfected with plasmid DNA expressing HBB sgRNA1 (250 ng) in GFP reporter cells grown in a 24-well plate. SaCas9 mRNA levels were compared to HBB sgRNA1 as a control by RT-qPCR. *** and ### indicate p<0.001 when SaCas90xMS2 was compared to SaCas91xMS2, and when SaCas91xMS2 was compared to SaCas9 containing more than one MS2 aptamer. Figure 9B shows the effect of MS2 aptamers on SaCas9 gene editing activity. GFP reporter cells were transfected in the same manner as in Figure 9A. Flow cytometry was performed 72 hours after transfection. *** indicates p<0.001 when SaCas90xMS2 is compared to SaCas9 containing at least one MS2 aptamer. Figure 9C shows the effect of aptamer number on LVLP gene editing activity. LVLP-containing supernatant (titer determined by p24 ELISA) was co-transduced into 2.5 × 10⁴ GFP reporter cells using 50 ng HBB sgRNA1-expressing IDLV. Flow cytometry was performed 72 hours after transduction. "a" shows that SaCas91xMS2LVLP with 45ng p24 obtained a significantly higher GFP positivity rate (p<0.001) than all other LVLPs at the same dosage; "b" shows that SaCas912xMS2LVLP with 45ng p24 obtained a significantly lower GFP positivity rate (p<0.001) than SaCas92xMS2 or SaCas93xMS2LVLP; and "c" shows that SaCas912xMS2LVLP with 15ng p24 obtained a significantly lower GFP positivity rate (p<0.05) than all other LVLPs at the same dosage. Each data point is the mean of three replicates. Figure 9D shows flow cytometry analysis of GFP-positive cells generated after transduction using various SaCas9-containing LVLPs.The indicated amount of SaCas9-containing LVLP was co-transduced into 2.5 × 10⁴ GFP reporter cells using 60 ng p24 HBB sgRNA1-expressing IDLV. Each point was the mean of the number of repeats shown (number in parentheses). * indicates p<0.05 when SaCas91xMS2-HBB 3'UTR LVLP is compared to any other particle of the same dosage; *** indicates p<0.001 when LVLP produced without ABP and aptamers is compared to any other particle of the same dosage. Figure 9E shows RT-qPCR comparison of SaCas9 mRNA copy numbers in different types of LVLP. RNA was purified from lentivirus or LVLP containing 30 ng p24. GFP lentivirus with 30 ng p24 was added to each sample as an experimental control. Copy numbers were compared to a standard lentiviral vector, which is known to contain 2 RNA genomes per LV particle. Shown are individual data points and mean ± SEM. ns, no significant difference; ***, p<0.001 compared to any other group. For Figures 9A, 9B, and 9E, Tukey's multiple comparison test was performed following the ANOVA analysis. For Figures 9C and 9D, Bonferroni post-tests were performed following the two-way ANOVA.

[0029] [Figure 10]Figures 10A-10E illustrate the characterization of LVLPs. Figure 10A shows transient expression of SaCas9 mRNA from LVLPs. 2.5 × 10⁴ HEK293T cells were transduced using SaCas9-expressing IDLV (35ng p24) or LVLP (35ng p24). SaCas9 mRNA levels were assayed at different time points. SaCas9 mRNA levels 24 hours after transduction were normalized by the housekeeping genes GAPDH and RPLP0 to obtain relative SaCas9 mRNA expression levels. Since no new mRNA was generated in LVLP transduced cells, subsequent normalization was not performed to avoid potential cell replication affecting the evaluation of mRNA degradation. *** indicates p<0.001 when SaCas91xMS2-HBB3'UTR is compared to other particles at the same time point. ### indicates p<0.001 when SaCas91xMS2 is compared to other particles at the same time point. Figure 10B shows the time course of SaCas9 mRNA levels from the same particle. mRNA levels in each particle were set to 1 24 hours after transduction. Shown are the mean ± SEM values ​​in the display repeat. * and ** indicate p<0.05 and p<0.01 when compared to other particles at the same time point. For Figures 10A and 10B, Bonferroni post-tests were performed following two-directional ANOVA. Figure 10C shows the Western blot analysis of the SaCas9 protein. The four lanes represent mock-transfected HREK293T cells (lane 1), GFP reporter cells co-transfected with 300 ng p24 Cas9 LVLP and 50 ng p24 HBB sgRNA1 IDLV (lane 2), and solubilates from HREK293T cells overexpressing Cas91xMS2 (lane 3) or Cas9 mRNA (lane 4) by transfecting 1.25 μg of DNA into 1.25 × 10⁵ cells. Very faint bands in LVLP-transfected cells are indicated by arrows. Figure 10D shows the processing of Gag precursors by HIV proteases. The width of the arrows is proportional to the processing rate at that site (1-5). Approximate sizes of p15-ABP fusion proteins are listed.Figure 10E shows the Western blot analysis of lentiviral proteins. 200 ng p24 GFP lentivirus, NC-MCP, and NC-PCP modified LVLP were analyzed.

[0030] [Figure 11] Figure 11 shows electron microscopy observations of NC-MCP and NC-PCP modified LVLP. Below the image are the mean ± SEM values, with the number of samples in parentheses. No statistically significant differences were observed between the groups.

[0031] [Figure 12-1]Figures 12A–F illustrate genome editing by SaCas9 LVLP. Figure 12A shows that SaCas9 LVLP efficiently generated indels at the perfectly matched HBB sgRNA1 target sequence. The most frequently observed sequences and their percentages from next-generation sequencing are listed. Red "T"s indicate mutations in the HBB that cause sickle cell disease. Figure 12B shows the frequency of indels at each location obtained from the sequencing data in Figure 12A. Figure 12C shows the indel in the IL2RG gene of HEK293T cells generated by SaCas9 LVLP. 2.5 × 10⁴ cells were co-transduced using 30 ng p24 SaCas91xMS2-HBB 3'UTR LVLP and 60 ng IDLV expressing IL2RG sgRNA. Figure 12D shows the indel in the IL2RG gene of lymphoblasts generated by 500ng p24 SaCas91xMS2-HBB 3'UTR LVLP (500ng p24 LVLP in 2 × 10⁵ cells). Figure 12E shows the indel rate at the wild-type HBB locus of GFP reporter cells. There was one mismatch between HBB sgRNA1 (highlighted in red) and the target sequence. Figure 12F shows the indel rate at the predicted off-target 8 of GFP reporter cells. Off-target 8 has one mismatch in HBB sgRNA1 (underlined), one DNA bulge (blue), and an atypical PAM (a "G" instead of a "T" at the last position, italicized). For Figures 12A-12F: Protospacer adjacent motifs (PAMs) or their complementary sequences are in green. For Figures 12A-12E: Target sequences are underlined. The vertical lines indicate complementarity between HBB sgRNA1 and the target. 3 to 4 million reads were obtained for each sample. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above.

[0032] [Figure 13]Figure 13 shows the relationship between the target sequence indel rate and the GFP positivity rate in GFP reporter cells. The four data points are derived from GFP reporter cells transduced using SaCas91xMS2LVLP (approximately 750 ng p24 per 1.25 × 10⁶ cells), SaCas9 AAV6 (10⁴ vg / cell), SaCas9 IDLV (approximately 750 ng p24 per 1.25 × 10⁶ cells), and SaCas91xMS2-HBB 3'UTR (30 ng p24 per 2 × 5 × 10⁴ cells). The target sequences in the GFP expression cassette were amplified and subjected to next-generation sequencing. The indel rate was plotted against the GFP positivity rate obtained by flow cytometry analysis.

[0033] [Figure 14]Figures 14A–14D illustrate the packaging of sgRNA in LVLPs. Figure 14A illustrates different Cas9 mRNA LVLPs (left) and Cas9 / sgRNA RNP LVLPs (right) as immature virions. Briefly, only one GAG ​​precursor and one mRNA or RNP are shown. ABPs can bind to aptamers as dimers. Figure 14B shows the location of MS2 aptamer insertions in the sgRNA scaffold. The original sgRNA is shown on the left (SEQ ID NO: 212). "N" indicates the guide sequence. Three locations examined for MS2 aptamer insertions are indicated by dashed black squares. The inserted sequences are shown on the right (SEQ ID NO: 213 (aptamer at the tetraloop) and SEQ ID NO: 214 (aptamer at the 3' end). Blue text within dashed blue squares indicates MS2 aptamers, and black text indicates addition linkers. Complementary ribonucleotides are shown by vertical lines, and atypical base pairings are shown by dots. Figure 14C shows the effect of MS2 aptamer position on the gene editing activity of RNPs containing modified sgRNAs. Plasmid DNA co-expressing SaCas9 mRNA and various modified HBB sgRNA1s was transfected into GFP reporter cells, and the GFP-positive percentage was determined by flow cytometry. Each data point represents one independent experiment. *, **, and *** are unmodified HBB Figure 14D shows p<0.05, p<0.01, and p<0.001 (Tukey's multiple comparison test following ANOVA) when compared to sgRNA1. The effect of MS2 aptamer position on the gene editing activity of modified sgRNA packaged in LVLP is shown. The indicated amounts of LVLP containing MS2-modified HBB sgRNA1 and SaCas9 protein (or mRNA) were used to transduce 2.5 × 10⁴ GFP reporter cells, and the GFP-positive percentage was determined by flow cytometry. Each data point is the mean of 3 replicates. ***, p<0.001, when HBB sgRNA1 tetraMS2 was compared to HBB sgRNA1 3'MS2; ###, p<0.001, when HBB sgRNA1 3'MS2 was compared to HBB sgRNA1 ST2 MS2.

[0034] [Figure 15] Figures 15A-15C show a comparison of aptamer / ABP pairs for packaging CRISPR / Cas9 RNA in LVLPs. Figure 15A shows the replacement of the tetraloop in SEQ ID NO: 215 with different aptamers for sgRNA packaging. The tetraloop (GAAA) sequence enclosed in a square was replaced with sequences containing or without a linker containing various aptamers (underlined) ((MS2 (SEQ ID NO: 216), com (SEQ ID NO: 217), PP7 (SEQ ID NO: 218), and BoxB (SEQ ID NO: 219)). Figure 15B shows a comparison of gene editing activity after the tetraloop has been replaced with different aptamers. Various amounts of plasmid DNA and aptamer-modified HBBs co-expressing SaCas9. shRNA1 was transfected into 1.25 × 10⁵ GFP reporter cells. 48 hours after transfection, GFP-positive cells were analyzed by flow cytometry. Each point was the mean of three replicates. *** indicates p<0.001 when sgRNAs without or with com-modification were compared to PP7, BoxB, or MS2 modified sgRNAs (Bonferroni post-test following ANOVA). Figure 15C shows a comparison of gene editing activity of LVLPs produced by different aptamer / ABP pairs. 400 μl of unenriched LVLP was used to transduce 2.5 × 10⁴ GFP reporter cells, and GFP positivity was determined by flow cytometry. Each point represents one independent assay. *** indicates p<0.001 between pairs of indications in Tukey's multiple comparison test following ANOVA analysis. ns, not significant.

[0035] [Figure 16]Figure 16 shows that LVLP generated GFP-positive cells in GFP reporter cells but not in HEK293T cells. 2.5 × 10⁴ cells were seeded in a 24-well plate and transduced using 150 ng p24 Cas9 / HBB sgRNA1 RNP LVLP. Cells were analyzed by fluorescence microscopy 48 hours after transduction.

[0036] [Figure 17] Figure 17 shows the analysis of NC-COM modification in particle assembly efficiency. Packaging plasmids with or without COM modification were used to package Cas9 / HBB sgRNA1 or GFP lentiviral vectors. Transfection was performed in 6-well plates; p24 was assayed in supernatant collected between 24 and 48 hours post-transfection. * indicates p<0.05 by Tukey's multiple comparison test following ANOVA.

[0037] [Figure 18]Figures 18A–18G illustrate that RNP is the primary contributor to gene editing activity. Figure 18A shows that HBB sgRNA1 expressed from transfected plasmid DNA is functional. The amount of DNA shown was transfected into 1.25 × 10⁵ GFP reporter cells, and the cells were analyzed by flow cytometry 48 hours after transfection. In the co-transfection experiment, the DNA amounts represent Cas9-expressed and sgRNA-expressing plasmid DNA, respectively. Each point was the average of three replicates. Figure 18B illustrates the importance of Cas9 co-packaging of sgRNA and com-aptamer modification for the gene editing activity of LVLPs. LVLPs packaged in the absence of Cas9 expression were inactive when co-transfected into GFP reporter cells using a functional Cas9-HBB-3'UTRMS2 mRNA LVLP. 2.5 × 10⁴ GFP reporter cells were transduced using Cas9 / HBB sgRNA1 LVLP (com-RNP LVLP), Cas9 / HBB sgRNA1 tetra-comLVLP (com+RNP LVLP), or co-transduced using 45 ng p24 of Cas9-HBB-3'UTRMS2 mRNA LVLP and varying amounts of HBB sgRNA1 tetra-comLVLP (com+sgRNA LVLP). GFP-positive cells were determined by flow cytometry 48 hours after transduction. Each point is the mean of three replicates. *** indicates p<0.001 when comparing the GFP positivity of cells treated with com+RNP LVLP to cells treated with similar amounts of other particles (Bonferroni post-test following ANOVA). Figure 18C shows that co-expression of Cas9 increased HBB sgRNA1 levels. Plasmids expressing only HBB sgRNA1 tetra-com (200 ng) and only SaCas9 (200 ng) were transfected into HEK293T cells individually or together, and sgRNA levels were compared by RT-qPCR. A GFP expression plasmid (50 ng) was co-transfected so that GFP expression could be used to normalize transfection efficiency.The total plasmid DNA was reduced to 450 ng with pCDNA3 plasmid DNA. * indicates p<0.05 when sgRNA levels without Cas9 co-expression are compared to those with Cas9 co-expression. Figure 18D shows Western blot analysis of Cas9 protein in isolated lentiviral vectors and LVLPs. 200 ng p24 GFP lentivirus (lane 1), NC-MCP modified Cas9-HBB-3'UTRMS2LVLP (without sgRNA, lane 2), NC-unmodified Cas9MS2LVLP (without sgRNA, lane 3), NC-COM modified Cas9 / HBB sgRNA1 tetra-comLVLP (lane 4), NC-COM modified Cas9 / IL2RG sgRNA1 tetra-comLVLP (lane 5), and NC-COM modified Cas9 / HBB sgRNA1 LVLP (sgRNA without tetra-com aptamer, lane 6) were loaded. Figure 18E shows that tetra-com modification of sgRNA increased the Cas9 protein content in LVLPs. Figure 18F shows that Cas9 protein in LVLPs with com-modified sgRNA is more surfactant-tolerant than Cas9 protein in LVLPs with unmodified sgRNA. Equal amounts of starting LVLP (200 ng of p24) were centrifuged through 1 ml of 10% sucrose with or without 0.5% Triton® X-100. For (Figures 18E and 18F), Cas9 levels were normalized by CA protein based on dosimetry analysis (IMAGE J). Figure 18G shows that sgRNA packaging in LVLPs is com-aptamer-dependent but not Cas9 protein-dependent. See Figure 18E for similar particle input (CA) evidence for RNA isolation. Each point indicates a single repeat. *** indicates p<0.001 between shown pairs in Tukey's multiple comparison test following ANOVA analysis.

[0038] [Figure 19] Figure 19 shows the standard curves when the same primer pair is used to amplify sgRNA DNA sequences with or without the com aptamer.

[0039] [Figure 20-1]Figures 20A-20E illustrate the efficiency and specificity of Cas9 / sgRNA RNP LVLP in gene editing. Figure 20A shows that Cas9 / sgRNA RNP LVLP has equivalent gene editing activity to Cas9 mRNA LVLP. For Cas9 mRNA LVLP, the particle amount is the sum of Cas9 mRNA LVLP and 60 ng p24 expressing HBB sgRNA1. Figure 20B shows the indel generated by Cas9 / IL2RG sgRNA1 RNP LVLP in the endogenous IL2RG target sequence. Protospacer adjacent motifs (PAMs) are grayed out, and the target sequence is underlined. Predicted cleavage sites are indicated by arrows. Dashed lines indicate deletions. Figure 20C shows the sequence of HBB sgRNA1, a Sickle mutant sequence that perfectly matches the gRNA, and an endogenous HBB sequence with one mismatch to the gRNA. Mutations that cause sickle cell disease are underlined. Figure 20D compares the on-target and off-target indel rates of Cas9 / HBB sgRNA1 RNP LVLP, including those from other delivery vehicles. The ratio of the on-target indel rate to the off-target indel rate ("on / off" ratio) is the result of dividing the on-target indel rate by the off-target indel rate. Cells were harvested 72 hours after treatment. For cell proliferation in 24-well plates, 1.25 × 10⁵ cells and 2.5 × 10⁴ cells were used for transfection and transduction, respectively. For RNP LVLPs, 150 ng p24 was used; for mRNA LVLPs, 45 ng p24 Cas9 mRNA LVLPs and 60 ng p24 HBB sgRNA1-expressing IDLVs were used; for LVs co-expressing Cas9 and HBB sgRNA1, 30 ng p24 was used; for IDLVs co-expressing Cas9 and HBB sgRNA1, 30 ng p24 was used; for Cas9 expressing AAV6 and HBB sgRNA1 expressing AAV6, 104 viral genomes / cells were used for each virus. On-target indel rates are all normalized to 100 for comparison. Original data are shown in Figure 22.Figure 20E shows that Cas9 RNP LVLP showed a faster effect than Cas9 mRNA LVLP. Transduction was performed into 2.5 × 10⁴ GFP reporter cells using either 50 ng p24 Cas9 / IL2RG sgRNA1 RNP LVLP or IDLV expressing 100 ng p24 Cas9 mRNA LVLP in addition to 100 ng p24 IL2RG sgRNA1, and the cells were incubated in IncuCyte for scanning. * indicates the time point at which RNP-treated cells showed a significantly higher (p<0.05) GFP-positive area / phase area than negative control or mRNA LVLP-treated cells. # indicates the time point at which mRNA LVLP-treated cells showed a significantly higher (p<0.05) GFP-positive area / phase area than negative control cells. The line shows the time difference until Cas9 mRNA LVLP-treated cells reached the same level of GFP-positive area / phase area as RNP-treated cells. [Figure 20-2] Same as above.

[0040] [Figure 21] Figure 21 shows indels generated by Cas9 / IL2RG RNP LVLP in lymphocytes. Gray nucleotides are PAMs. Underlined nucleotides are target sequences. Underlined italicized nucleotides are insertions.

[0041] [Figure 22] Figures 22A and 22B show that Cas9 / sgRNA RNP LVLP promoted homologous recombination in the presence of a donor template delivered by IDLV. Figure 22A shows the design of the donor template and the PCR strategy for detecting homologous recombination. Figure 22B shows the detection of homologous recombination events by NGS. PAM is green. Target sequences are underlined. Inserted IL2RG cDNA is orange. The original start codon and the start codon for the inserted cDNA are red. In cDNA, lowercase letters indicate that the sequence is identical to the original cDNA sequence, while uppercase letters indicate that the sequence is different from the original but encodes the same protein. [Modes for carrying out the invention]

[0042] definition As used herein and in the appended claims, unless otherwise expressly indicated, the singular forms “a,” “an,” and “the” include plural references.

[0043] The terms “nucleic acid” or “nucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in either single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise noted, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994). The term nucleic acid is used synonymously with genes, cDNA, and mRNA encoded by genes.

[0044] The term "gene" may refer to a segment of DNA involved in the production or coding of polypeptide chains. This may include the regions before and after the coding region (leaders and trailers), as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" may refer to a segment of DNA involved in the production or coding of non-coding RNA, such as rRNA, tRNA, guide RNA, or microRNA.

[0045] "Treating" means any indication of success in treating, restoring, or preventing a disease, condition, or disorder, including any objective or subjective parameters, such as relief, remission, reduction, or making the disease state more tolerable to the patient, slowing the rate of degeneration or decline, or making the final stage of degeneration less debilitating. Treatment or restoring symptoms may be based on objective or subjective parameters, including the results of a physician's examination. Accordingly, the term "treating" includes the administration of the compounds or agents of this disclosure to prevent, delay, alleviate, stop, or inhibit the onset of symptoms or conditions associated with a disease, condition, or disorder as described herein. The term "therapeutic effect" means the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject. "Treating" or "treating" using the methods of this disclosure may include preventing the onset of symptoms, inhibiting the symptoms of the disease or disorder (slowing or stopping their onset), providing relief from the symptoms or side effects of the disease (including symptomatic treatment), and alleviating the symptoms of the disease (causing regression) in subjects who are at increased risk of a disease or disorder associated with a disease or disorder as described herein, but who have not yet experienced or shown symptoms. Treatment may be preventive (to prevent or delay the onset of the disease or to prevent the manifestation of its clinical or asymptomatic symptoms) or therapeutic suppression or reduction of symptoms after manifestation. The term "treatment," as used herein, includes preventive (e.g., prophylactic), curative, or symptomatic treatment.

[0046] A “promoter” is defined as one or more nucleic acid regulatory sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the transcription start site, for example, a TATA element in the case of a polymerase type II promoter. The promoter may also include a distal enhancer or repressor element, which may be located several thousand base pairs away from the transcription start site, as needed.

[0047] The terms “polypeptide,” “peptide,” and “protein” are used synonymously herein to refer to polymers of amino acid residues. All three terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. As used herein, the term encompasses full-length proteins, cleaved proteins and their fragments, and amino acid chains in which amino acid residues are linked by covalent peptide bonds.

[0048] As used herein, the terms “complementary” or “complementarity” refer to the specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U) and G and C.

[0049] Throughout this text, "subject" refers to an individual. For example, a subject is a mammal, such as a primate, and more specifically, a human. Non-human primates are also subjects. The term "subject" includes domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Veterinary and medical uses and formulations are thus intended herein. This term does not indicate a specific age or sex. Therefore, adult and neonatal subjects, whether male or female, are considered subjects. Where used herein, patient or subject may be used synonymously and can refer to a subject suffering from a disease or disorder.

[0050] An expression cassette is a nucleic acid construct constructed by recombination or synthesis using a set of designated nucleic acid elements that enable the transcription of a specific polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette contains the polynucleotide to be transcribed, operably ligated to a promoter, followed by a transcription termination signal sequence. An expression cassette may or may not contain designated regulatory sequences, such as the 5' or 3' untranslated region derived from the human globin gene.

[0051] A "reporter gene" encodes a protein that is readily detectable by its biochemical characteristics, such as enzymatic activity or chemifluorescence properties. These reporter proteins can be used as selection markers. One specific example of such a reporter is green fluorescent protein. The fluorescence produced by this protein can be detected using various commercially available fluorescence detection systems. Other reporters can be detected by staining. A reporter can also be an enzyme that produces a detectable signal when in contact with a suitable substrate. A reporter can be an enzyme that catalyzes the formation of a detectable product. Suitable enzymes include, but are not limited to, proteases, nucleases, lipases, phosphatases, and hydrolases. A reporter may encode an enzyme that allows the substrate to be substantially impermeable to the eukaryotic plasma membrane, thus enabling robust control of signal formation. Specific examples of suitable reporter genes encoding enzymes include, but are not limited to, CAT (chloramphenicol acetyltransferase; Alton and Vapnek (1979) Nature 282: 864-869), luciferase (lux), β-galactosidase, LacZ, β-glucuronidase, and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182: 231-238 and Hall et al. (1983) J. Mol. Appl. Gen. 2: 101) is cited, and the full text of each of them is incorporated herein by reference. Other suitable reporters include those that encode specific epitopes that can be detected using labeled antibodies that specifically recognize the epitope.

[0052] The "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of bacteria and archaea. The CRISPR / Cas system includes subtypes I, II, and III.

[0053] The CRISPR / Cas system classification, as described by Makarova, et al. (Nat Rev Microbiol. 2015 Nov; 13(11):722-36), defines five types and 16 subtypes based on shared features and evolutionary similarities. These are grouped into two major classes based on the structure of the effector complex that cleaves genomic DNA. The Type II CRISPR / Cas system was the first to be used for genome engineering, followed by Type V in 2015. The wild-type Type II CRISPR / Cas system, in complex with guide RNA, utilizes the RNA-mediated nuclease Cas protein or homolog (referred herein to as “CRISPR-associated endonuclease”) to recognize and cleave foreign nucleic acids. The Cas9 protein also uses activating RNA (also called transactivating or tracrRNA). Depending on the type of CRISPR-associated endonuclease used together, guide RNAs, or guide RNAs having the activity of either guide RNA or both guide RNA and activating RNA, are also known in the art. In some cases, such dual-active guide RNAs are called single guide RNAs (sgRNAs). Synthetic guide RNAs that do not contain an activating RNA sequence may also be called sgRNAs. In this disclosure, the terms sgRNA and gRNA are used synonymously to refer to RNA molecules that complex with CRISPR-associated endonucleases and localize the ribonucleoprotein complex to a target DNA sequence. Methods and compositions for controlling the inhibition and / or activation of the transcription of target genes, populations of target genes (e.g., controlling the transcriptome or a portion thereof) are described, for example, in Cell. 2014 Oct 23;159(3):647-61, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0054] As used herein, “activity” refers to the ability to bind to a target gene element, in relation to CRISPR / Cas activity, CRISPR-related endonuclease activity, sgRNA activity, sgRNA:CRISPR-related endonuclease nuclease activity, etc. Typically, activity also refers to the ability of the sgRNA:CRISPR-related endonuclease nuclease complex to produce double-strand breaks in a target genomic region. In some cases, activity may refer to the ability to modulate transcription in or near a target genomic region. Such activity can be measured by various methods known in the art. For example, the expression, activity, or level of a gene containing or adjacent to a target genomic region may be measured. In another example, the production of insertions and deletions (indels) in the cell's genome at a target genomic region may be measured.

[0055] As used herein, the term “edit” in relation to editing the genome of a cell refers to inducing a structural change in the sequence of a genome in a target genome region. For example, editing may take the form of inserting a nucleotide sequence into the genome of a cell or deleting a nucleotide sequence from the genome of a cell. The nucleotide sequence may encode a polypeptide or a fragment thereof. Such editing may be carried out by inducing a double-strand break or a pair of single-strand nicks in the opposite strand and adjacent target genome regions within the target genome region. Methods for inducing single-strand or double-strand breaks in or within a target genome region include the use of a CRISPR-associated endonuclease nuclease domain and a guide RNA or a pair of guide RNAs directed to the target genome region.

[0056] As used herein, non-homologous end joining (NHEJ) refers to a cellular process in which a cleaved or nicked end of a DNA strand can be directly ligated without the need for a homologous template nucleic acid. NHEJ can result in the addition, deletion, substitution, or combination thereof of one or more nucleotides at the site to be repaired.

[0057] As used herein, the term homologous recombination repair (HDR) refers to a cellular process in which a cleaved or nicked end of a DNA strand is repaired by polymerization from a homologous template nucleic acid. Thus, the original sequence is replaced by the template sequence. The homologous template nucleic acid may be provided by a homologous sequence from another location in the genome (sister chromatids on the same or different chromosomes, homologous chromosomes, or repeat regions). Alternatively, an exogenous template nucleic acid may be introduced to obtain a specific HDR-inducible alteration of the sequence at a target site. In this method, a specific mutation may be introduced at the cleavage site.

[0058] As used herein, “target template sequence” refers to a DNA oligonucleotide that can be used by a cell as a template for an HDR. A target template sequence may be a single-stranded DNA template or a double-stranded DNA template. Generally, a target template sequence has at least one region homologous to a target site (genomic target sequence or target genomic region) in the cell’s genome. In some examples, a target template sequence has two homologous regions adjacent to a region containing a heterologous sequence to be inserted into a target cleavage site (genomic target sequence or target genomic region) in the cell’s genome.

[0059] As used herein, terms such as “ribonucleoprotein complex” and “RNP” refer to complexes between CRISPR-related endonucleases, e.g., Cas9 protein and crRNA (e.g., guide RNA or single guide RNA), Cas9 protein and transactivating crRNA (tracrRNA), Cas9 protein and guide RNA, or combinations thereof (e.g., complexes containing Cas9 protein, tracrRNA, and crRNA guide). Detailed explanation

[0060] The following description enumerates various aspects and embodiments of the composition and method. No particular embodiment defines the scope of the composition and method. Rather, the embodiments simply provide a range of unlimited examples of various compositions and methods that fall within the scope of the disclosed composition and method. The description should be understood from the perspective of those skilled in the art, and therefore does not necessarily include information readily known to those skilled in the art.

[0061] Compositions, systems, methods for production, and procedures for efficiently delivering CRISPR / Cas system components to eukaryotic cells using viral particles are provided herein. For example, components, systems, methods for production, and procedures for efficiently delivering CRISPR-related endonuclease mRNA or RNP to cells via lentivirus-like particles are provided. CRISPR-related endonucleases produced by the systems described herein are functional for gene editing in eukaryotic cells. CRISPR-related endonuclease mRNA is delivered into eukaryotic cells, has a limited half-life, and reduces the risk of off-target mediated mutagenesis. Delivery of RNP to eukaryotic cells enables efficient delivery, for example, to cells that are difficult to transfect, such as primary cells, while reducing off-target effects.

[0062] Lentivirus-like particles contain modified viral proteins, which are fusion proteins with aptamer-binding proteins. These modified viral proteins may be structural or non-structural. In particular, modified viral proteins may be lentiviral regulatory proteins (VPRs and NEFs), nucleocapsid (NC) proteins, or matrix (MA) proteins, which have aptamer-binding proteins fused to them. Lentivirus-like particles also contain non-viral nucleic acid sequences, specifically, one or both of the following: CRISPR-associated endonuclease coding sequences (CRISPR-associated endonuclease mRNA) or guide RNA (gRNA). As shown in Figures 1A and 1B, these CRISPR / Cas system components are packaged within the lentivirus-like particles and delivered into eukaryotic cells. In some cases, gRNA is delivered using lentivirus-like particles. For example, in some cases, the CRISPR-associated endonuclease coding sequence and gRNA may be packaged together within the lentivirus-like particle. Alternatively, the CRISPR-related endonuclease coding sequence and gRNA may be packaged within a separate lentivirus-like particle. Furthermore, other delivery mechanisms may be used to deliver the gRNA into eukaryotic cells (e.g., lentivirus, adenovirus, adeno-associated virus, plasmid, gRNA transfection, or electroporation).

[0063] Nonviral nucleic acid sequences can be modified by adding aptamer sequences to which aptamer-binding proteins, fused to modified viral proteins, specifically bind. In some embodiments, the aptamer sequence is attached to a nucleic acid encoding CRISPR-related endonuclease mRNA. The interaction between the aptamer sequence and the aptamer-binding protein fused to the modified viral protein facilitates the packaging of endonuclease mRNA into lentiviral particles. Figure 14A illustrates the packaging of CRISPR-related endonuclease mRNA in lentiviral particles via the interaction between an aptamer attached to Cas mRNA and an aptamer-binding protein fused to a viral protein. In some embodiments, the aptamer sequence is attached to or inserted into a gRNA sequence. When an aptamer sequence attached to or inserted into a gRNA sequence interacts with an aptamer-binding protein, the gRNA sequence, which is complexed with CRISPR-related endonuclease (RNP), is packaged into lentiviral particles. Figure 14B illustrates the packaging of RNPs (i.e., CRISPR-related endonucleases complexed with gRNA) in lentiviral particles via the association of aptamers attached to gRNA and aptamer-binding proteins fused to viral proteins.

[0064] The presence of a viral fusion protein can increase the packaging of nonviral nucleic acid sequences or RNPs within lentivirus-like particles. In some cases, the addition of an aptamer sequence to a nonviral nucleic acid sequence, for example, the addition of an aptamer sequence to a nucleic acid sequence encoding CRISPR-related endonuclease mRNA, can further increase the amount of RNA packaged. In other cases, the nonviral nucleic acid sequence includes a gRNA sequence and a sequence encoding CRISPR-related endonuclease, and the addition of an aptamer sequence to the gRNA sequence can further increase the amount of RNP packaged. This disclosure provides lentivirus-like particles as described above, other viral particle components, plasmids for constructing such lentivirus-like particles, methods and systems for constructing lentivirus-like particles using such plasmids, and methods for modifying genomic target sequences in cells using lentivirus-like particles. I. Introduction

[0065] The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, which forms the adaptive immune system in bacteria, has been modified for genome engineering. The engineered CRISPR / Cas system contains two components: a guide RNA (gRNA, also called a single guide RNA (sgRNA)) and a CRISPR-associated endonuclease. The gRNA is a short synthetic RNA consisting of a scaffold sequence necessary for binding to the CRISPR-associated endonuclease and a user-defined spacer of approximately 20 nucleotides that defines the genomic target to be modified. Therefore, the genomic target of the CRISPR-associated endonuclease can be altered by simply changing the target sequence present in the gRNA. Originally used to knock out target genes in various cell types and organisms, CRISPR has been extended through modifications to various CRISPR-related endonucleases to selectively activate / repress target genes, purify specific regions of DNA, image DNA in living cells, and precisely edit DNA and RNA. When homologous recombination repair (HDR) of a genomic target is desired, for example, when the goal is to correct a mutated genomic sequence, the system includes a target template sequence that provides the desired sequence to be introduced into the genome in place of the mutated genomic sequence. When non-homologous end joining (NHEJ) is the repair mechanism used to repair breaks in genomic DNA, no target template sequence is used, and the repair generally results in a deletion or insertion at the repair site. This mechanism is useful when the desired outcome from gene editing is to inactivate and / or impair the function of a genomic sequence, or to restore the reading frame of a gene disrupted by a deletion or insertion.

[0066] Various mammalian expression systems have been used to deliver the CRISPR / Cas system into cells. These include lentiviral transduction, adeno-associated virus (AAV) transduction, mammalian expression vectors, direct delivery of CRISPR-related endonuclease mRNA and gRNA, and direct delivery of CRISPR-related endonuclease / gRNA ribonucleoprotein complexes.

[0067] In lentiviral systems, CRISPR-related endonucleases and gRNAs may be present in a single lentiviral vector or in separate lentiviral vectors. Viral vectors may contain a reporter gene (such as GFP) to identify and enrich positive cells. Lentiviral vectors also often contain selection markers to create stable cell lines. A safety characteristic of lentiviral systems is that the components necessary to produce infectious viral particles (virions) are generally distributed across multiple plasmids. Packaging plasmids and envelope plasmids encode components of the viral capsid and envelope, and are used together with a transfer plasmid encoding the viral genome and one or both of the CRISPR-related endonucleases or gRNAs. These plasmids are simultaneously transfected into cells (such as human embryonic kidney cells), the cells are incubated, and the supernatant containing the viral particles is collected. These viral particles can then be used to transduce cells of interest. A portion of the lentiviral vector genome can then be incorporated into the target cell genome, modulating the target gene and its expression. However, the use of lentiviral vector systems to deliver CRISPR-related endonucleases carries the potential to induce oncogenic changes, infectious changes, and other transformative changes in infected cells.

[0068] Different types of lentiviral vector systems have been developed to improve the safety and efficacy of lentiviral vector systems. Second-generation lentiviral systems contain a single packaging plasmid encoding the Gag, Pol, Rev, and Tat genes. Transgene expression is driven by a weak promoter, the genomic 5'LTR, without an internal promoter, and requires the presence of Tat to activate expression. Third-generation systems improve upon the safety of the second-generation system in two ways. First, the packaging system is divided into two packaging plasmids: one encoding Rev, and the other encoding Gag and Pol. Second, Tat has been excluded from the third-generation system, and transgene expression from this promoter no longer depends on Tat transactivation. Third-generation transfer plasmids can be packaged by either the second or third-generation packaging system. While second and third-generation systems address concerns associated with the unintended generation of reproducible viruses, these systems are still prone to mutagenesis and off-target effects in transduced cells.

[0069] CRISPR-related endonucleases and / or gRNAs can be inserted into AAV transfer vectors and used with the AAV system to generate AAV particles. The packaging limit for AAV particles is only about 4.5 kb, which limits the size of the CRISPR-related endonucleases and gRNAs that can be used.

[0070] When mammalian expression vectors are used, a heterologous promoter is used to drive CRISPR-related endonuclease expression. The promoter can be constitutive or inductive. The U6 promoter is often used for gRNA. Expression vectors may contain a reporter gene (such as green fluorescent protein; GFP) for identifying and enriching positive cells, or a selection marker for creating a stable cell line. Mammalian expression vectors may be used for transient or stable expression of CRISPR-related endonucleases and / or gRNA in mammalian cell lines that can be transfected with high efficiency.

[0071] CRISPR-related endonuclease mRNA and gRNA (synthesized from plasmids using in vitro transcription reactions) can be delivered to target cells using microinjection or electroporation. Similarly, purified CRISPR-related endonuclease protein and in vitro transcribed gRNA can be combined in vitro to form a ribonucleoprotein complex, which is then delivered to cells using cationic lipids. Both direct delivery methods result in transient expression of CRISPR components, as the expression of CRISPR-related endonuclease mRNA or protein and / or gRNA decreases as they are degraded within the cell.

[0072] Aspects of this disclosure include modified lentiviral vector systems and components, which in some cases may also include one or more lentiviral components, AAV components, or mammalian expression vectors. In some cases, the modified lentiviral vector systems and components provided are modified to exclude all or a substantial portion of the lentiviral genome. Furthermore, lentiviral-like particles produced from the modified systems and components have reduced risks of generating infectious particles in transduced cells, as well as risks of causing mutagenesis and off-target effects. II. Plasmid Components

[0073] This disclosure provides various plasmid compositions, including modified lentiviral packaging plasmids, modified lentiviral transfer plasmids, and mammalian expression plasmids. A. Modified viral protein plasmid

[0074] One aspect of the present disclosure is a plasmid comprising a polynucleotide sequence encoding a modified lentiviral protein, which is a fusion protein of a lentiviral protein fused to an aptamer-binding protein. In connection with the present disclosure, the modified viral protein may be structural or non-structural. In some cases, the modified viral protein is a structural protein and may be provided by a lentiviral packaging plasmid. In some cases, the modified viral protein is a non-structural protein and may be provided by a mammalian expression vector. Exemplary structural proteins include lentiviral nucleocapsid (NC) proteins and matrix (MA) proteins. Exemplary non-structural proteins include viral protein R (VPR) and negative regulators (NEF).

[0075] In some embodiments, the modified viral protein is a nucleocapsid (NC) protein. In other embodiments, the modified viral protein is a matrix (MA) protein. Both the NC and MA proteins are encoded by the lentiviral Gag gene. In some cases, the coding sequence of the viral protein may be one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. In some cases, the amino acid sequence of the viral protein may be one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some cases, the lentiviral packaging plasmid contains a sequence encoding at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, operably linked to a eukaryotic cell promoter. In some cases, if the viral protein is a NEF, the polypeptide may contain three mutations that enhance packaging in the viral capsid, e.g., the following substitution mutations: G3C, V153L, and E177G.

[0076] The polynucleotide encoding the modified lentiviral protein contains an aptamer-binding protein (ABP) coding sequence. In some cases, the ABP coding sequence is located at the 5' or 3' end of the viral protein coding sequence. In some cases, the ABP coding sequence may be inserted into the viral protein coding sequence so that the encoded ABP fuses with the viral protein. The ABP coding sequence may be inserted in-frame at an internal location within the viral protein coding sequence. When positioned in-frame at an internal location near the 5' or 3' end of the viral protein coding sequence, the ABP coding sequence is positioned so as not to disrupt processing sequences, such as those described in J. Virol. 65(2):922-30 (1991) and Biochimica et Biophysica Acta - Biomembranes 1614(1):62-72 (2003), the full text of which is incorporated herein by reference. For example, the Gag nucleotide sequence encodes, among other things, the NC coding sequence and the MA coding sequence, and the Gag precursor protein is processed into separate mature viral proteins by proteolytic cleavage. In-frame insertion of the ABP coding sequence does not disrupt the nucleotides encoding the processing sequence for cleavage by proteolysis. In some cases, nucleotides in the viral protein coding sequence may be replaced by the ABP protein coding sequence. In some cases, a linker sequence encoding 3-6 amino acids may be positioned between or adjacent to the ABP coding sequence to help facilitate proper folding of the protein domain during expression.

[0077] In one example, the modified viral protein is NC, and the ABP coding sequence is inserted at the 5' or 3' end of the NC coding sequence. In another example, the modified viral protein is NC, and the ABP coding sequence is inserted before or after one of the zinc finger (ZF) domains. For example, the ABP coding sequence may be inserted after the last codon of the second ZF (ZF2) domain. In yet another example, the ABP coding sequence may be inserted before the first codon of the ZF2 domain. In yet another example, the ABP coding sequence may be inserted before the first codon of the first ZF (ZF1) domain. In yet another example, the ABP coding sequence may be inserted after the last codon of the first ZF (ZF1) domain. In some cases, the ABP coding sequence is inserted into the NC coding sequence in a way that does not disrupt the highly positive stretch of amino acids in the NC protein.

[0078] In another example, the modified viral protein is MA, and the ABP coding sequence is inserted at the 5' or 3' end of the MA coding sequence. In yet another example, the ABP coding sequence is inserted in-frame at an internal position within the MA coding sequence. In some cases, nucleotides in the MA coding sequence may be replaced by those in the ABP protein coding sequence. For example, nucleotides encoding amino acids 44–132 of the MA protein may be replaced by those in the ABP coding sequence. In yet another example, the ABP coding sequence is inserted before the codon encoding amino acid 44 of the MA protein. In yet another example, the ABP coding sequence is inserted after the codon encoding amino acid 132 of the MA protein.

[0079] In another example, the modified viral protein is VPR, and the ABP coding sequence is inserted into the 5' or 3' end of the VPR coding sequence. In one example, the ABP coding sequence is inserted into the 5' end of the VPR coding sequence.

[0080] In another example, the modified viral protein is NEF, and the ABP coding sequence is inserted into the 5' or 3' end of the NEF coding sequence. In one example, the ABP coding sequence is inserted into the 3' end of the NEF coding sequence.

[0081] In one embodiment, a lentiviral packaging plasmid is provided comprising a eukaryotic cell promoter operably ligated to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, and one or both of the NC coding sequence or the MA coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence. In some cases, the packaging plasmid does not encode a functional integrase protein. In some cases, the lentiviral packaging plasmid includes at least one nonviral ABP nucleotide sequence immediately downstream of a second zinc finger domain of the NC coding sequence. In such cases, the NC coding sequence may include two functional zinc finger protein domains and a functional native protease processing sequence. Retention of the zinc finger protein domains and native protease processing sequences ensures that the modified NC fusion protein expressed from the lentiviral packaging plasmid is correctly processed into a functional protein. In some cases, lentiviral packaging plasmids contain at least one nonviral ABP nucleotide sequence within the MA coding sequence.

[0082] In some cases, a plasmid may encode one or more viral proteins, including two or more fused aptamer-binding proteins. In certain cases, the Gag nucleotide sequence of a lentiviral packaging plasmid may include an NC coding sequence and an MA coding sequence, where one or both of the NC coding sequence or the MA coding sequence include a first nonviral ABP nucleotide sequence and a second nonviral ABP nucleotide sequence. Both the first and second nonviral ABP nucleotide sequences may encode the same ABP. Alternatively, the first and second nonviral ABP nucleotide sequences may encode different ABPs. In some cases, the Gag nucleotide sequence of a lentiviral packaging plasmid may include an NC coding sequence containing at least one first nonviral ABP nucleotide sequence and an MA coding sequence containing at least one second nonviral ABP nucleotide sequence. Both the at least one first nonviral ABP nucleotide sequence and at least one second nonviral ABP nucleotide sequence may encode the same ABP. Alternatively, at least one first nonviral ABP nucleotide sequence and at least one second nonviral ABP nucleotide sequence encode different ABPs.

[0083] In certain cases, a mammalian expression vector may encode a VPR-coding sequence or a NEF-coding sequence, which includes a first nonviral ABP nucleotide sequence and a second nonviral ABP nucleotide sequence. Both the first and second nonviral ABP nucleotide sequences may encode the same ABP. Alternatively, the first and second nonviral ABP nucleotide sequences may encode different ABPs.

[0084] Nonviral aptamer-binding protein (ABP) nucleotide sequences encode polypeptide sequences that bind to RNA aptamer sequences. Several nonviral ABPs are suitable for use in this disclosure. Particularly suitable ABPs include bacteriophage RNA-binding proteins that specifically bind to RNA sequences that form a stem-loop structure called RNA aptamer sequences. Exemplary nonviral aptamer-binding proteins include MS2 coat protein, PP7 coat protein, lambda N peptide, and COM (Control of mom) protein. The lambda N peptide may be amino acids 1-22 of the lambda N protein, which is the RNA-binding domain of the protein. In some cases, ABPs bind to their aptamers as dimers. Information on these ABPs and the aptamer sequences to which they bind is provided in Table 1 below. In some embodiments, at least one nonviral ABP nucleotide sequence encodes a polypeptide having the sequence shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16. In some embodiments, at least one nonviral ABP nucleotide sequence includes one of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. Table 1. Aptamer-binding proteins and corresponding aptamer sequences [Table 1]

[0085] As discussed above, lentiviral packaging plasmids can encode various lentiviral proteins. In some cases, a packaging plasmid containing only the Gag nucleotide sequence (gene), Pol nucleotide sequence (gene), Rev nucleotide sequence (gene), and Tat nucleotide sequence (gene) may be called a second-generation packaging plasmid. In some cases, a packaging plasmid containing only the Gag nucleotide sequence (gene) and Pol nucleotide sequence (gene) may be called a third-generation packaging plasmid. In some cases, a lentiviral packaging plasmid may encode only the proteins necessary for the viral shell (capsid). In some cases, the coding sequences of one or more lentiviral proteins may be excluded in whole or in part from the packaging plasmid. For example, in some cases, the packaging plasmid contains only the Gag nucleotide sequence (gene). In another example, a lentiviral packaging plasmid may contain deletions of all or part of the Pol nucleotide sequence (gene). In yet another example, a lentiviral packaging plasmid may contain deletions of all or part of the integrase (Int) coding sequence. In another example, a lentiviral packaging plasmid may contain deletions of all or part of the reverse transcriptase (RT) coding sequence.

[0086] A characteristic of the lentiviral packaging plasmids provided herein is that they may not encode a functional integrase protein. When a packaging plasmid does not encode a functional integrase protein and is used in the systems and methods described herein, the risk of nucleic acid molecules held by lentiviral-like particles produced using these packaging plasmids being incorporated into the genome of transduced eukaryotic cells is substantially reduced. In some cases, lentiviral packaging plasmids contain an integrase-coding sequence having an integrase-inactivating mutation. For example, the integrase-inactivating mutation may be a mutation from aspartic acid to valine at amino acid position 64 of the integrase protein encoded by the integrase-coding sequence (D64V). In some cases, lentiviral packaging plasmids contain deletions of all or part of the integrase-coding sequence.

[0087] Lentiviral packaging plasmids contain a eukaryotic cell promoter operably ligated to a Gag nucleotide sequence. Mammalian expression plasmids contain a eukaryotic cell promoter operably ligated to a VPR coding sequence or a NEF coding sequence. In some cases, the eukaryotic cell promoter is an RNA polymerase II promoter. The RNA polymerase II promoter sequence is selected from a mammalian species. For example, the promoter sequence may be selected from, to name a few, human, bovine, sheep, buffalo, pig, or mouse. In some cases, the RNA polymerase II promoter sequence is a CMV, FE1α, or SV40 sequence. In some cases, the RNA polymerase II sequence is a modified RNA polymerase II sequence. For example, RNA polymerase II sequences having at least 80%, 85%, 90%, 95%, or 99% identity to a wild-type RNA polymerase II promoter sequence from any mammalian species may be used in the constructs provided herein. Those skilled in the art will readily understand how to determine the identity of two polypeptides or nucleic acids. For example, identity can be calculated after aligning two sequences so that their identity is at its highest level. Another method for calculating identity can be performed using publicly available algorithms. For example, the algorithm in Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970) can be used to perform optimal alignment of sequences for comparison. In some cases, eukaryotic promoters are inducible promoters.

[0088] The coding sequence transcribed from the RNA pol II promoter contains poly(A) signaling and transcriptional terminator sequences downstream of the coding sequence. Commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) contain the sequence motif AAUAAA, which promotes both polyadenylation and termination. The role of a terminator, a sequence-based element, is to define the end of a transcription unit (such as a gene) and initiate the process of releasing newly synthesized RNA from the transcription mechanism. Terminators are found downstream of the gene to be transcribed and typically occur immediately after any 3' regulatory element, such as polyadenylation or poly(A) signaling.

[0089] In some cases, lentiviral packaging plasmids may contain expression cassettes. In some cases, mammalian expression plasmids may contain expression cassettes. B. CRISPR system component plasmids

[0090] Mammalian expression plasmids used to deliver CRISPR component coding sequences into mammalian cells used to produce the lentivirus-like particles of this disclosure are provided herein. Once introduced into mammalian cells together with the lentivirus packaging plasmids or mammalian expression plasmids described in Section IIA, these mammalian expression plasmids act as templates for producing nonviral CRISPR component RNA molecules that are packaged in the lentivirus-like particles. In some cases, the mammalian expression plasmid is a lentivirus transfer vector. Lentivirus transfer vectors generally encode a viral genome in addition to the exogenous gene of interest. In some cases, any mammalian expression plasmid may be suitable for the expression of CRISPR component coding sequences in cells used to produce lentivirus-like particles.

[0091] One aspect of this disclosure is a mammalian expression plasmid comprising a coding sequence for one or more CRISPR components. In some cases, the CRISPR component coding sequence comprises at least one aptamer sequence coding sequence. When the CRISPR component coding sequence is transcribed from a mammalian expression vector, the aptamer sequence is similarly transcribed. The resulting transcribed RNA molecule comprises the CRISPR component RNA sequence and at least one aptamer sequence. As will be discussed in more detail below, the CRISPR component coding sequence may be a CRISPR-associated endonuclease coding sequence, a gRNA coding sequence, or both. In some cases, the CRISPR component coding sequence may be a CRISPR-associated endonuclease coding sequence. In some cases, the CRISPR-associated endonuclease coding sequence may contain at least one aptamer sequence following the stop codon of the CRISPR-associated endonuclease coding sequence but before any poly(A) signal and / or transcription terminator. In some cases, the CRISPR component coding sequence may be a gRNA coding sequence. In some cases, the gRNA coding sequence includes at least one aptamer coding sequence. In some cases, at least one aptamer coding sequence may be located at the 5' or 3' end of the gRNA. In some cases, at least one aptamer coding sequence may be inserted into an internal location within the gRNA, for example, into one or more of the loops formed in the folded gRNA. For example, if the gRNA is for the SaCas9 protein, at least one aptamer coding sequence may be located at the tetraloop, stemloop 2, or 3' end of the gRNA. In some cases, the aptamer sequence is inserted as a tetraloop.

[0092] In some cases, the aptamer coding sequence attached to or inserted into the gRNA sequence is selected from the group consisting of aptamers that bind to the MS2 coat protein sequence, aptamers that bind to the PP7 coat protein, aptamers that bind to the lambda N peptide, and aptamers that bind to the COM protein. In some cases, the aptamer coding sequence is the MS2 coat protein aptamer sequence, e.g., SEQ ID NO: 17 (RNA) or SEQ ID NO: 18 (DNA). In some cases, the aptamer coding sequence is the PP7 coat protein aptamer sequence, e.g., SEQ ID NO: 19 (RNA) or SEQ ID NO: 20 (DNA). In some cases, the aptamer coding sequence is the lambda N peptide aptamer sequence, e.g., SEQ ID NO: 21 (RNA) or SEQ ID NO: 22 (DNA). In some cases, the aptamer coding sequence is the COM protein aptamer sequence, e.g., SEQ ID NO: 17 (RNA) or SEQ ID NO: 18 (DNA).

[0093] In some cases, the mammalian expression plasmid contains a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence. In some cases, each of the CRISPR-associated endonuclease coding sequence and the gRNA coding sequence may contain at least one aptamer coding sequence. In some cases, the CRISPR-associated endonuclease coding sequence contains at least one aptamer coding sequence downstream of it. In some cases, the gRNA coding sequence contains at least one aptamer coding sequence. In some cases, a 1-30 nucleotide spacer may be positioned between the CRISPR component coding sequence and at least one aptamer coding sequence, or adjacent to at least one aptamer coding sequence.

[0094] One aspect of the present disclosure is a mammalian expression plasmid comprising the coding sequences of one or more CRISPR components. In some cases, the CRISPR component coding sequences comprise at least one aptamer sequence coding sequence.

[0095] Throughout, sgRNAs interact with CRISPR-associated endonucleases (CRISPR site-specific nucleases) and specifically bind to or hybridize with target nucleic acids (genomic target sequences) in the cell's genome, resulting in a single guide RNA sequence in which the sgRNA and CRISPR-associated endonuclease coexist with the target nucleic acid in the cell's genome. Each sgRNA contains a DNA targeting sequence or protospacer sequence approximately 10–50 nucleotides in length that specifically binds to or hybridizes with a target DNA sequence in the genome. For example, the DNA targeting sequence may be approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. For example, the DNA targeting sequence may be approximately 15-30 nucleotides, approximately 15-25 nucleotides, approximately 10-25 nucleotides, or approximately 18-23 nucleotides. In one example, the DNA targeting sequence is approximately 20 nucleotides. In some embodiments, the sgRNA includes a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the sgRNA does not include a tracrRNA sequence.

[0096] Generally, DNA targeting sequences are designed to complement (e.g., perfectly complement) or substantially complement (e.g., have 1 to 4 mismatches) the target DNA sequence. In some cases, DNA targeting sequences may incorporate fluctuations or degenerate bases to bind to multiple gene elements. In some cases, the 19 nucleotides at the 3' or 5' end of the binding region are perfectly complementary to the target gene element(s). In some cases, the binding region may be modified to increase stability. For example, non-native nucleotides may be incorporated to increase RNA resistance to degradation. In some cases, the binding region may be modified or designed to avoid or reduce secondary structure formation in the binding region. In some cases, the binding region may be designed to optimize the GC content. In some cases, the GC content is preferably between about 40% and about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some cases, the binding region may be selected to begin with a sequence that promotes efficient transcription of sgRNA. For example, the binding region may begin with a G nucleotide at its 5' end. In some cases, the binding region may contain modified nucleotides, such as, but not limited to, methylated or phosphorylated nucleotides.

[0097] As used herein, the terms “complementary” or “complementarity” refer to base pairing between nucleotides or nucleic acids, for example, base pairing between an sgRNA and a target sequence, though not limited to these. Complementary nucleotides are generally A and T (or A and U) and G and C. Guide RNAs described herein may include sequences that are fully complementary or substantially complementary to a genomic sequence (e.g., having 1–4 mismatches), such as DNA targeting sequences.

[0098] The sgRNA includes a constant region of sgRNA that interacts with or binds to a CRISPR-related endonuclease. In the constructs provided herein, the constant region of the sgRNA may be about 75–250 nucleotides in length. In some examples, the constant region is a modified constant region containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide substitutions in the stem, stem-loop, hairpin, inter-hairpin region, and / or the constant region nexus. In some cases, a modified constant region having at least 80%, 85%, 90%, or 95% of the activity of the natural or wild-type sgRNA constant region from which the modified constant region is derived may be used in the constructs described herein. In particular, the modification should not be made at nucleotides that directly interact with a CRISPR-related endonuclease, such as the Cas9 polypeptide, or at nucleotides that are important to the secondary structure of the constant region.

[0001] CRISPR-related endonucleases encoded by mammalian expression plasmids as described herein include RNA-induced site-specific nucleases. Examples include, but are not limited to, nucleases present in any bacterial species encoding a type II or type V CRISPR / Cas system. For example, but are not limited to, CRISPR-related endonucleases may be Cas9 polypeptide (type II) or Cpf1 polypeptide (type V). See, for example, Abudayyeh et al., Science 2016 August 5; 353(6299):aaf5573; Fonfara et al. Nature 532: 517-521 (2016), and Zetsche et al., Cell 163(3): p.759-771, 22 October 2015. Throughout this text, the term “Cas9 polypeptide” means the Cas9 protein or a fragment or derivative thereof identified in any bacterial species encoding a type II CRISPR / Cas system. For example, see Makarova et al. Nature Reviews, Microbiology, 9: 467-477 (2011), the full text of which, including supplementary information, is incorporated herein by reference. CRISPR-related endonucleases, such as Cas9 and Cas9 homologs, are found in a variety of eubacteria, including, but not limited to, the following taxonomic groups of bacteria: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein (SpCas9). Another exemplary Cas9 protein is the Staphylococcus aureus Cas9 protein (SaCas9).Additional Cas9 proteins and their homologs are, for example, Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou,. The Cas9 nuclease domain is described in *Proc Natl Acad Sci USA. 2013 Sep 24;110(39):15644-9*, *Sampson et al., Nature. 2013 May 9;497(7448):254-7*, and *Jinek, et al., Science. 2012 Aug 17;337(6096):816-21*. The Cas9 nuclease domain can be optimized for efficient activity or enhanced stability in host cells. Other CRISPR-related endonucleases include Cpf1 (e.g., Zetsche et al., Cell, (See Volume 163, Issue 3, pp. 759-771, 22 October 2015) and its homologs.

[0099] Full-length Cas9 is an endonuclease containing a recognition domain and two nuclease domains (HNH and RuvC, respectively) that create double-strand breaks in DNA sequences. In the amino acid sequence of Cas9, HNH is linearly continuous, while RuvC is divided into three regions: one to the left of the recognition domain and the other two adjacent to the HNH domain on the right side of the recognition domain. Cas9 is targeted to genomic sites in cells by interacting with a guide RNA that hybridizes with a 20-nucleotide DNA sequence immediately preceding the NGG motif recognized by Cas9. This results in a double-strand break in the cell's genomic DNA. In some cases, Cas9 nucleases that require an NGG protospacer adjacent motif (PAM) immediately 3' of the region targeted by the guide RNA can be utilized. As another example, a Cas9 protein with orthogonal PAM motif requirements can be used to target sequences that do not have an adjacent NGG PAM sequence. Examples of Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, those described in Esvelt et al., Nature Methods 10: 1116-1121 (2013).

[0100] In some embodiments, the Cas9 protein may be in an active endonuclease form so that a double-strand break is introduced into the target nucleic acid when it is bound to the target nucleic acid as part of a complex with guide RNA or as part of a complex with a DNA template. The double-strand break can be repaired by an NHEJ, which introduces random mutations, or by an HDR, which introduces specific mutations. Various Cas9 nucleases can be used in the manner described herein. For example, a Cas9 nuclease requiring an NGG protospacer adjacent motif (PAM) immediately 3' of the region targeted by the guide RNA, such as SpCas9, can be used. Such a Cas9 nuclease can be targeted to any region of the genome containing an NGG sequence. In another example, a Cas9 nuclease requiring an NNGRRT or NNGRR(N)PAM immediately 3' of the region targeted by the guide RNA, such as SaCas9, can be used. In yet another example, a Cas9 protein having an orthogonal PAM motif requirement can be used to target sequences that do not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, those described in Nature Methods 10, 1116-1121 (2013) and those described in Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015.

[0101] In some cases, the Cas9 protein is a nickase, and as a result, when it binds to a target nucleic acid as part of a complex with a guide RNA, a single-strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally different guide RNA, can target two proximal sites in the target genomic region and thus introduce a pair of proximal single-strand breaks into the target genomic region. Since off-target effects are generally likely to result in a single nick that is repaired without damage by the base excision repair mechanism, a pair of nickases can provide enhanced specificity. Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations.

[0102] In some cases, CRISPR-related endonucleases are site-specific nucleases. For example, a CRISPR-related endonuclease may be a dCas9 polypeptide. As used throughout this disclosure, the dCas9 polypeptide is an inactivated or nuclease-dead Cas9 (dCas9) modified to inactivate Cas9 nuclease activity. Modifications include, but are not limited to, altering one or more amino acids to inactivate nuclease activity or a nuclease domain. For example, but are not limited to, D10A and H840A mutations can be produced in Cas9 derived from Streptococcus pyogenes to inactivate Cas9 nuclease activity. Other modifications include removing all or part of the nuclease domain of Cas9 so that no sequence exhibiting nuclease activity is present in Cas9. Therefore, dCas9 may involve a modified polypeptide sequence to inactivate nuclease activity, or the removal of one or more polypeptide sequences that inactivate nuclease activity. dCas9 may have inactivated nuclease activity but retain the ability to bind to DNA. Thus, dCas9 may contain the polypeptide sequence(s) necessary for DNA binding but a modified nuclease sequence, or it may lack the nuclease sequence involved in nuclease activity. It is understood that similar modifications can be made to inactivate nuclease activity in other site-specific nucleases, such as Cpf1 or C2c2. In some examples, the dCas9 protein is a full-length Cas9 sequence derived from S. pyogenes that lacks the polypeptide sequences of the RuvC nuclease domain and / or HNH nuclease domain, but retains DNA binding function. In other examples, the dCas9 protein sequence lacks the RuvC nuclease domain and / or the HNH nuclease domain and has at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to the Cas9 polypeptide sequence that retains DNA-binding function.

[0103] In some cases, an inactivated CRISPR-related endonuclease is linked to the effector protein. If necessary, a site-specific nuclease is linked to the effector protein via a peptide linker. The linker can be approximately 2 to 25 amino acids long. The effector protein may be a transcription regulatory protein or its active fragment. The transcription regulatory protein may be a transcription activator or transcription repressor protein, or the protein domain of an activator or inhibitor protein. Examples of transcription activators, but not limited to them, include VP16, VP48, VP64, VP192, MyoD, E2A, CREB, KMT2A, NF-KB(p65AD), NFAT, TET1, p300Core, and p53. Examples of transcription inhibitors, but not limited to them, include KRAB, MXI1, SID4X, LSD1, and DNMT3A / B. Effector proteins may also be epigenome editors, such as histone acetyltransferases, histone demethylases, or DNA methylases. Effector proteins or their active fragments may be sequentially operably ligated to the amino or carboxyl terminus of a CRISPR-related endonuclease. If necessary, two or more activated effector proteins or their active domains may be operably ligated to the amino or carboxyl terminus of a CRISPR-related endonuclease. If necessary, two or more repressor effector proteins or their active domains may be sequentially operably ligated to the amino or carboxyl terminus of a CRISPR-related endonuclease. If necessary, effector proteins do not necessarily need to be covalently ligated to a CRISPR-related endonuclease, but may associate, conjugate, or otherwise connect to the nuclease.

[0104] In some embodiments, the CRISPR-associated endonuclease is the Cpf1 polypeptide. The Cpf1 protein is a type II, type V CRISPR / Cas system protein. Cpf1 is a smaller and simpler endonuclease than Cas9 (e.g., spCas9). The Cpf1 protein has a RuvC-like endonuclease domain similar to that of Cas9, but without the HNH endonuclease domain. The N-terminal domain of Cpf1 also lacks the alpha-helix recognition lobe found in the Cas9 protein. When cleaving DNA, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4 or 5 nucleotide overhang. The Cpf1 protein does not require tracrRNA; rather, the Cpf1 protein functions with crRNA alone. In relation to this disclosure, if the CRISPR-associated endonuclease is the Cpf1 protein, the sgRNA does not contain a tracr sequence. The sgRNA used with the Cpf1 protein may contain only the crRNA sequence (constant region). In some cases, the TTTN or TTN may be located immediately 5' of the region targeted by the guide RNA. Cpf1 proteins requiring a PAM (where "N" is a nucleic acid base, depending on the species) can be utilized. Known Cpf1 proteins and their derivatives may be used in connection with this disclosure. For example, in some cases the CRISPR-associated endonuclease is FnCpf1p and the PAM is 5'TTN (where N is A / C / G or T). In some cases the CRISPR-associated endonuclease is PaCpf1p and the PAM is 5'TTTV (where V is A / C or G). In certain specific cases the CRISPR-associated endonuclease is FnCpf1p and the PAM is 5'TTN (where N is A / C / G or T), and the PAM is located upstream of the 5' end of the protospacer. In certain cases, the CRISPR-associated endonuclease is FnCpf1p, and the PAM is 5'CTA, located upstream of the 5' end of the protospacer or at the target locus. In one example, the CRISPR-associated endonuclease is AsCpf1p, and the PAM is 5'TTTN.

[0105] Mammalian expression plasmids contain a eukaryotic cell promoter operably ligated to a nonviral nucleic acid sequence. In some cases, the eukaryotic cell promoter is an RNA polymerase II promoter, and the nonviral nucleic acid sequence is a CRISPR-associated endonuclease coding sequence. In other cases, the eukaryotic cell promoter is an RNA polymerase III promoter, and the nonviral nucleic acid sequence is a gRNA coding sequence. In some cases, the nonviral nucleic acid sequence contains both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, with the RNA polymerase II promoter operably ligated to the CRISPR-associated endonuclease coding sequence and the RNA polymerase III promoter operably ligated to the gRNA coding sequence.

[0106] The RNA polymerase II promoter sequence is selected from mammalian species. The RNA polymerase III promoter sequence is selected from mammalian species. For example, these promoter sequences may be selected from, to name a few, human, bovine, sheep, buffalo, pig, or mouse. In some cases, the RNA polymerase II promoter sequence is the CMV, FE1α, or SV40 sequence. In some cases, the RNA polymerase III promoter sequence is the U6 or H1 sequence. In some cases, the RNA polymerase II sequence is a modified RNA polymerase II sequence. For example, RNA polymerase II sequences having at least 80%, 85%, 90%, 95%, or 99% identity to wild-type RNA polymerase II promoter sequences derived from any mammalian species may be used in the constructs provided herein. In some cases, the RNA polymerase III sequence is a modified RNA polymerase III sequence. For example, RNA polymerase III sequences having at least 80%, 85%, 90%, 95%, or 99% identity to wild-type RNA polymerase III promoter sequences derived from any mammalian species may be used in the constructs provided herein. Those skilled in the art will readily understand how to determine the identity of two polypeptides or nucleic acids. For example, identity can be calculated after aligning two sequences to their highest level of identity. Another method for calculating identity can be carried out by publicly available algorithms. For example, the algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970) can be used to perform optimal alignment of sequences for comparison. In some cases, eukaryotic cell promoters are inducible promoters.

[0107] The coding sequence transcribed from the RNA pol II promoter contains poly(A) signaling and transcriptional terminator sequences downstream of the coding sequence. Commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) contain the sequence motif AAUAAA, which promotes both polyadenylation and termination. The coding sequence transcribed from the RNA pol III promoter contains a simple run of T residues downstream of the coding sequence as a terminator sequence. The role of a terminator, a sequence-based element, is to define the end of a transcription unit (such as a gene) and initiate the process of releasing newly synthesized RNA from the transcription mechanism. Terminators are found downstream of the gene to be transcribed and typically occur immediately after any 3' regulatory element, such as polyadenylation or poly(A) signaling.

[0108] In some cases, a mammalian expression vector includes at least one aptamer-coding sequence that encodes an aptamer sequence to which an aptamer-binding protein (ABP) specifically binds. In relation to this disclosure, the aptamer sequence is an RNA sequence that forms a tertiary loop structure to which ABP specifically binds. ABP is an RNA-binding protein or an RNA-binding protein domain. Suitable aptamer-coding sequences include polynucleotide sequences that encode known bacteriophage aptamer sequences. Exemplary aptamer-coding sequences include those that encode the aptamer sequences provided above in Table 1. In some cases, the aptamer is bound to a dimer of ABP. These aptamer sequences are RNA sequences to which bacteriophage proteins are known to specifically bind. In some situations, at least one aptamer-coding sequence encodes an aptamer sequence to which ABP specifically binds, selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda-N RNA-binding domain, or COM protein.

[0109] In some cases, a mammalian expression vector contains a CRISPR component coding sequence that includes one aptamer coding sequence downstream. In other cases, a CRISPR component coding sequence may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 aptamer coding sequences. For example, in some cases, a CRISPR component coding sequence may contain two aptamer coding sequences in tandem. If a mammalian expression vector contains both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, either or both may contain one or more aptamer coding sequences. In one example, if a vector contains both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, the CRISPR-associated endonuclease coding sequence may contain one or more aptamer coding sequences. In another example, if a vector contains both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, the gRNA coding sequence may contain one or more aptamer coding sequences. In another example, if a vector contains both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, both the CRISPR-associated endonuclease coding sequence and the gRNA coding sequence may each contain one or more aptamer coding sequences. If a vector contains both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, both contain one or more aptamer coding sequences, and one or more aptamer coding sequences in the CRISPR-associated endonuclease coding sequence and one or more aptamer coding sequences in the gRNA coding sequence may be the same aptamer coding sequence or different aptamer coding sequences. In some cases, a mammalian expression plasmid contains a CRISPR component coding sequence, and the CRISPR component coding sequence does not contain an aptamer coding sequence.

[0110] In some cases, a mammalian expression plasmid includes a CRISPR-associated endonuclease coding sequence containing at least one first aptamer coding sequence and a gRNA coding sequence containing at least one second aptamer coding sequence. In one example, at least one first aptamer coding sequence and at least one second coding aptamer sequence are the same aptamer coding sequence. In some cases, at least one first aptamer coding sequence codes for an aptamer sequence to which a first ABP specifically binds, at least one second aptamer coding sequence codes for an aptamer sequence to which a second ABP specifically binds, and at least one first aptamer coding sequence and at least one second aptamer coding sequence code for aptamer sequences to which different first and second ABPs bind. For example, at least one first aptamer coding sequence and at least one second aptamer coding sequence may encode an aptamer sequence to which an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA-binding domain, and COM protein specifically binds. In another example, at least one first aptamer coding sequence and at least one second aptamer coding sequence may encode an aptamer sequence to which different ABPs selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA-binding domain, and COM protein each specifically bind.

[0111] In some cases, mammalian expression plasmids may also include at least one polynucleotide sequence encoding an RNA stabilization sequence, which is located downstream of the CRISPR component coding sequence, or, if located downstream of the CRISPR component coding sequence, downstream of the aptamer coding sequence. The polynucleotide sequence encoding the RNA stabilization sequence is transcribed downstream of the CRISPR / Cas system component coding sequence and stabilizes the longevity of the transcribed RNA sequence. In one example, the polynucleotide sequence encoding the RNA stabilization sequence is located downstream of the CRISPR-associated endonuclease coding sequence. In another example, the polynucleotide sequence encoding the RNA stabilization sequence is located downstream of the gRNA coding sequence. An exemplary RNA stabilization sequence is the 3'UTR sequence of the human betaglobin gene, as shown in SEQ ID NO: 25 (DNA) and SEQ ID NO: 26 (RNA). Other RNA stabilization sequences are described in Hayashi, T. et al., Developmental Dynamics 239(7):2034-2040 (2010) and Newbury, S. et al., Cell 48(2):297-310 (1987). In some cases, a 1-30 nucleotide spacer may be placed between the CRISPR component coding sequence and at least one polynucleotide sequence encoding the RNA stabilization sequence.

[0112] In some cases, mammalian expression plasmids may contain an expression cassette. In some cases, mammalian expression plasmids may also contain a reporter gene. TEAS.system

[0113] Another aspect of the present disclosure is a lentiviral packaging system. Such a system includes the lentiviral packaging plasmid and mammalian expression plasmid described in the present disclosure. These systems are useful in providing components for introduction into mammalian cells to produce the lentiviral-like particles described in the present disclosure.

[0114] In some cases, the system comprises a lentiviral packaging plasmid containing a eukaryotic cell promoter operably linked to a Gag nucleotide sequence, the Gag nucleotide sequence comprising a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, and either or both of the NC coding sequence or the MA coding sequence comprising at least one nonviral aptamer-binding protein (ABP) nucleotide sequence, and the packaging plasmid does not encode a functional integrase protein. The lentiviral packaging plasmid may have any of the configurations described above in Section II.A. The system may comprise a second-generation packaging plasmid or a third-generation packaging plasmid, or modified versions thereof. In some cases, the packaging plasmid comprises a Gag nucleotide sequence as described above, and further comprises a Rev nucleotide sequence and a Tat nucleotide sequence. In other cases, the system comprises a first packaging plasmid containing a Gag nucleotide sequence as described above and a second packaging plasmid containing a Rev nucleotide sequence. In each packaging plasmid, the viral protein-coding sequence is operably ligated to a eukaryotic cell promoter, for example, individually or to a single promoter for multiple protein-coding sequences.

[0115] In other cases, the system comprises a mammalian expression plasmid containing a eukaryotic cell promoter operably ligated to an NEF coding sequence or a VPR coding sequence, the NEF coding sequence or VPR coding sequence containing at least one nonviral ABP nucleotide sequence. The mammalian expression plasmid may have any of the configurations described above in Section II.A. The system may comprise a second-generation packaging plasmid or a third-generation packaging plasmid or a modified version thereof. In some cases, the packaging plasmid contains a Gag nucleotide sequence, a Rev nucleotide sequence, and a Tat nucleotide sequence. In other cases, the system comprises a first packaging plasmid containing a Gag nucleotide sequence and a second packaging plasmid containing a Rev nucleotide sequence.

[0116] The system may also include at least one mammalian expression plasmid containing a eukaryotic cell promoter operably linked to a nonviral nucleic acid sequence, the nonviral nucleic acid sequence containing a CRISPR-related endonuclease coding sequence, a guide RNA (gRNA) coding sequence, or both a CRISPR-related endonuclease coding sequence and a gRNA coding sequence. The mammalian expression plasmid may have any of the configurations described above in Section II.B. In some cases, the system includes a mammalian expression plasmid containing a eukaryotic cell promoter operably linked to a CRISPR-related endonuclease coding sequence and a eukaryotic cell promoter operably linked to a gRNA coding sequence. In other cases, the system includes a first mammalian expression plasmid containing a eukaryotic cell promoter operably linked to a CRISPR-related endonuclease coding sequence and a second mammalian expression plasmid containing a eukaryotic cell promoter operably linked to a gRNA coding sequence. In some cases, the nonviral nucleic acid in at least one mammalian expression plasmid does not contain an aptamer sequence. In other cases, the nonviral nucleic acid in at least one mammalian expression plasmid contains at least one aptamer sequence.

[0117] The system may also include an envelope plasmid having an envelope coding sequence that encodes a viral envelope glycoprotein. For example, the Env nucleotide sequence may encode VSV-G. The envelope coding sequence is operably ligated to a eukaryotic promoter. In some cases, the eukaryotic promoter is the RNA pol II promoter. Appropriate eukaryotic promoters are described in Section II.

[0118] Kits containing components of the systems described herein are also provided herein. In some embodiments, the kits include one or more plasmids described herein. IV. Production of lentivirus-like particles

[0119] Methods for producing lentivirus-like particles using plasmids and systems described herein are provided herein.

[0120] In one embodiment, a method for producing lentiviral particles is provided, comprising the steps of transfecting a plurality of eukaryotic cells with a packaging plasmid, at least one mammalian expression plasmid, and an envelope plasmid as described above in Sections II and III, and culturing the transfected eukaryotic cells for a time sufficient to produce lentiviral particles. The method may use a second-generation packaging plasmid or a third-generation packaging plasmid. In some cases, first and second packaging plasmids as described in Section III are used. The plurality of eukaryotic cells may be mammalian cells. For example, the mammalian cells may be human embryonic kidney cells or another suitable mammalian expression cell line.

[0121] In some cases, fusion with viral protein-coding sequences, such as NC-coding sequences, MA-coding sequences, or both, does not interfere with viral particle assembly when a plasmid encoding a viral fusion protein, at least one mammalian expression plasmid encoding a nonviral nucleotide sequence, and an envelope plasmid are transfected into eukaryotic cells, with one or more ABP nucleotide sequences interfering with viral particle assembly. In some cases, lentiviral packaging plasmids encoding NC-ABP fusion proteins, MA-ABP fusion proteins, or both NC-ABP and MA-ABP fusion proteins do not interfere with viral particle assembly. In some cases, mammalian expression plasmids encoding either NEF-ABP fusion proteins or VPR-ABP fusion proteins do not interfere with viral particle assembly.

[0122] Depending on the plasmid used to transfect eukaryotic cells, different lentivirus-like particles are produced by the methods provided. In some cases, eukaryotic cells are transfected with a mammalian expression plasmid containing a CRISPR-associated endonuclease coding sequence. Cells transfected with this plasmid produce lentivirus-like particles containing CRISPR-associated endonuclease mRNA. In some cases, eukaryotic cells are transfected with a mammalian expression plasmid containing a gRNA coding sequence. Cells transfected with this plasmid produce lentivirus-like particles containing gRNA. In some cases, cells may be transfected with a first mammalian expression plasmid containing a CRISPR-associated endonuclease coding sequence and a second mammalian expression plasmid containing a gRNA coding sequence. Cells transfected with these plasmids produce lentivirus-like particles containing both CRISPR mRNA and gRNA. In some cases, cells may be transfected with mammalian expression plasmids containing both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence. Cells transfected with this plasmid produce lentivirus-like particles containing both CRISPR mRNA and gRNA.

[0123] Generally, eukaryotic cells are simultaneously transfected with a plasmid, the cells are allowed to incubate, and then the supernatant containing the virus-like particles is collected. The culture medium may be changed after transfection and before incubation. The collected virus-like particles are stored and can be centrifuged to concentrate the particles. Routine cell transfection methods are used to produce lentiviral particles. Crude or concentrated virus-like particles can then be used to transduce cells of interest. Viral titers can also be determined using known protocols.

[0124] A characteristic of the methods provided is the amount of nonviral RNA molecules that can be packaged within lentivirus-like particles. In natural lentiviruses, the viral shell (capsid) contains two copies of a single-stranded RNA genome. For example, a wild-type HIV-1 virus particle contains two copies of a single-stranded RNA genome of approximately 9.7 kb. The methods provided by this disclosure can package a large number of nonviral RNA molecules within the viral shell. In some cases, the methods can produce lentivirus-like particles containing up to 100 nonviral RNA molecules. For example, lentivirus-like particles may contain approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nonviral RNA molecules. For example, the method may produce particles containing 5–25 nonviral RNA molecules, 25–50 nonviral RNA molecules, 50–75 nonviral RNA molecules, or 75–100 nonviral RNA molecules. For example, the method may produce particles containing approximately 50–100 nonviral RNA molecules. In some cases, the method may produce particles containing up to 100 copies of CRISPR-related endonuclease mRNA (e.g., SaCas9 mRNA). In some cases, the method may produce lentivirus-like particles containing several copies of CRISPR-related endonuclease mRNA, gRNA, or a combination thereof. In some cases, the mammalian expression plasmid used in the method may contain a nonviral nucleic acid sequence containing at least one aptamer coding sequence. In other cases, the mammalian expression plasmid used in the method may contain a nonviral nucleic acid sequence that does not contain at least one aptamer sequence. In some cases, mammalian expression plasmids do not require an aptamer coding sequence downstream of a nonviral nucleic acid sequence that generates a lentiviral particle containing several copies of a nonviral RNA molecule.

[0125] In some cases, mammalian expression plasmids contain a non-viral nucleic acid sequence encoding CRISPR-related endonuclease mRNA, and the aptamer-coding sequence is attached to the nucleic acid encoding the CRISPR-related endonuclease mRNA. The interaction between the aptamer sequence and the aptamer-binding protein in the viral fusion proteins described herein facilitates the packaging of CRISPR-related endonuclease mRNA into lentiviral particles. In some cases, mammalian expression plasmids contain non-viral nucleic acid sequences encoding CRISPR-related endonuclease mRNA and gRNA, and the aptamer-coding sequence is attached to or inserted into the gRNA. The interaction between the aptamer sequence attached to or inserted into the gRNA in the viral fusion proteins described herein and the aptamer-binding protein facilitates the packaging of gRNA, which has been complexed with CRISPR-related endonuclease (RNP), into lentiviral particles. V. Lentivirus-like particles

[0126] In another embodiment, lentivirus-like particles are provided. The lentivirus-like particles contain a modified lentiviral protein which is a fusion protein in which at least one aptamer-binding protein is fused to one or more viral proteins. In relation to this disclosure, the modified viral protein may be structural or non-structural. Exemplary structural proteins include lentiviral nucleocapsid (NC) proteins and matrix (MA) proteins. Exemplary non-structural proteins include viral protein R (VPR) and negative regulator (NEF). In some cases, the particles contain an NC protein and an MA protein, one or both of which contain a fusion protein fused to at least one non-viral aptamer-binding protein (ABP). The NC protein of the particle may have two functional zinc finger protein domains. In particular, retention of a second NC zinc finger domain may preserve the efficiency of viral assembly and budding. In some cases, the particles contain a fusion protein comprising a VPR protein or an NEF protein, the VPR protein or NEF protein being fused with at least one nonviral ABP. The particles also contain at least one nonviral RNA sequence, the nonviral RNA sequence comprising CRISPR-related endonuclease mRNA, guide RNA (gRNA), or both CRISPR-related endonuclease mRNA and gRNA. In some cases, the lentivirus-like particles do not contain a functional integrase protein. These virus-like particles are useful for transduction into target eukaryotic cells.

[0127] The particles may contain a viral fusion protein comprising one or more ABPs. In some cases, the particles may contain an NC protein, an MA protein, or both, with one or both of the NC or MA proteins fused to one or more nonviral ABPs. In some cases, the lentivirus-like particles contain an NC protein fused to at least one nonviral ABP. In some cases, the lentivirus-like particles contain an MA protein fused to at least one nonviral ABP. In some cases, the lentivirus-like particles may contain both an NC protein and an MA protein, with one or both of the NC or MA proteins fused to two nonviral ABP proteins, a first nonviral ABP and a second nonviral ABP fused to the C-terminus of the first nonviral ABP (i.e., in tandem). In certain specific cases, the particles may contain one or both of the NC or MA proteins fused to a first nonviral ABP and a second nonviral ABP. In some cases, lentivirus-like particles contain a VPR protein or NEF protein, which is fused with one or more nonviral ABPs. In some cases, lentivirus-like particles contain two nonviral ABPs: a first nonviral ABP and a VPR protein or NEF protein fused to a second nonviral ABP (i.e., in tandem) fused to the C-terminus of the first nonviral ABP. In some cases, lentivirus-like particles contain a VPR protein or NEF protein fused to both the first nonviral ABP and the second nonviral ABP. Both the first and second nonviral ABPs may be the same ABP. Alternatively, the first and second nonviral ABPs may be different ABPs. In some cases, the lentivirus-like particle may contain an NC protein having at least one first nonviral ABP fused to an MA protein having at least one second nonviral ABP fused to its C-terminus.At least one first nonviral ABP and at least one second nonviral ABP are both the same ABP. Or, at least one first nonviral ABP protein and at least one second nonviral ABP may be different ABPs. Both the first nonviral ABP and the second nonviral ABP may be the same ABP. Or, the first nonviral ABP and the second nonviral ABP may be different ABPs.

[0128] Nonviral ABPs are polypeptide sequences that bind to RNA aptamer sequences. Several nonviral ABPs are suitable for use in this disclosure. Particularly suitable ABPs include bacteriophage RNA-binding proteins that specifically bind to known RNA aptamer sequences, which are RNA sequences that form a stem-loop structure. Exemplary nonviral aptamer-binding proteins include MS2 coat protein, PP7 coat protein, lambda N peptide, and COM (Control of mom) protein. The lambda N peptide may be amino acids 1-22 of the lambda N protein, which is the RNA-binding domain of the protein. Information regarding these ABPs and the aptamer sequences to which they bind is provided above in Table 1. In some embodiments, at least one nonviral ABP protein is a polypeptide having the sequence shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.

[0129] Lentivirus-like particles may contain various lentiviral proteins. However, in some cases, lentivirus-like particles do not contain all types of proteins or nucleic acids found in natural lentiviruses. In some cases, particles may contain NC, MA, CA, SP1, SP2, P6, POL, ENV, TAT, REV, VIF, VPU, VPR, and / or NEF proteins, or any of their derivatives, combinations, or parts. In some cases, particles may contain NC, MA, CA, SP1, SP2, P6, and POL. In some cases, lentivirus-like particles may contain only these proteins that form the viral shell (capsid). In some cases, one or more lentiviral proteins may be excluded from lentivirus-like particles, in whole or in part. For example, in some cases, lentivirus-like particles may not contain the POL protein, or they may contain non-functional versions of the POL protein, such as POL proteins with inactivation site mutations or inactivation end cleavage. In another example, lentivirus-like particles may not contain integrase proteins, but may contain non-functional versions of integrase proteins, such as integrase proteins with inactivation site mutations or inactivation end cleavage. For example, lentivirus-like particles may contain a non-functional integrase protein with an aspartate-to-valine mutation at amino acid position 64 (D64V). In yet another example, lentivirus-like particles may not contain reverse transcriptase proteins, but may contain non-functional versions of reverse transcriptase proteins, such as reverse transcriptase proteins with inactivation site mutations or inactivation end cleavage.

[0130] In some cases, lentivirus-like particles contain at least one nonviral RNA molecule. The nonviral RNA molecule is either at least one component of the CRISPR / Cas system or encodes a component of the CRISPR / Cas system. In some cases, the nonviral RNA molecule may be mRNA encoding a CRISPR-related endonuclease. Suitable CRISPR-related endonucleases are discussed throughout this disclosure. In one example, the nonviral RNA molecule may be mRNA encoding the Cas9 protein or a derivative thereof. In another example, the nonviral RNA molecule may be mRNA encoding the Cpf1 protein or a derivative thereof. In some cases, the nonviral RNA molecule may include gRNA. Characteristics of suitable gRNAs are discussed throughout this disclosure. The gRNA generally contains a DNA targeting sequence and a constant region that interacts with a CRISPR-related endonuclease. In some cases, the gRNA may contain a transactivating crRNA (tracrRNA) sequence. For example, the gRNA may contain tracrRNA that should be used with the Cas9 protein or a derivative thereof. In other cases, the gRNA does not contain a tracrRNA sequence. For example, the gRNA may not contain a tracrRNA sequence that should be used with the Cpf1 protein or derivative. In some cases, the lentivirus-like particles contain both mRNA and gRNA encoding a CRISPR-related endonuclease. In some cases, the particles may contain either mRNA or gRNA encoding a CRISPR-related endonuclease.

[0131] In some cases, at least one nonviral RNA molecule may have at least one aptamer sequence located at its 3' end. The aptamer sequence is a sequence known to be specifically bound to an aptamer-binding protein (ABP) fused to a viral protein as described above. As discussed above, the aptamer sequence is an RNA sequence that forms a tertiary loop structure to which ABP specifically binds. Suitable aptamer sequences include known bacteriophage aptamer sequences. Exemplary aptamer sequences are provided above in Table 1. These aptamer sequences are RNA sequences to which bacteriophage proteins specifically bind. In some situations, at least one aptamer sequence is specifically bound to an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA-binding domain, or COM protein.

[0132] In some cases, the nonviral RNA molecule may be CRISPR-associated endonuclease mRNA. In some cases, the CRISPR-associated endonuclease mRNA may contain at least one aptamer sequence at its 3' end. In some cases, the nonviral RNA molecule may be a gRNA coding sequence. In some cases, the gRNA contains at least one aptamer sequence. In some cases, at least one aptamer sequence may be located at the 5' or 3' end of the gRNA. In some cases, at least one aptamer sequence may be inserted into an internal position within the gRNA, for example, into one or more loops formed in the folded gRNA. For example, if the gRNA is for the SaCas9 protein, at least one aptamer sequence may be located at the tetraloop, stemloop 2, or 3' end of the gRNA. In some cases, at least one nonviral RNA molecule may include CRISPR-associated endonuclease mRNA and gRNA. In some cases, each of the CRISPR-associated endonuclease mRNA and gRNA may contain at least one aptamer coding sequence. In some cases, the CRISPR-associated endonuclease mRNA contains at least one aptamer sequence at its 3' end. In some cases, the gRNA contains at least one aptamer sequence. In some cases, a 1-30 ribonucleotide spacer may be positioned between the CRISPR-associated endonuclease mRNA or gRNA and at least one aptamer sequence, or adjacent to at least one aptamer sequence. In certain specific cases, at least one aptamer sequence does not interfere with the transduction of lentiviral-like particles in eukaryotic cells. For example, at least one nonviral ABP fused to one or more of the NC protein, MA protein, VPR protein, or NEF protein may not interfere with the transduction of lentiviral-like particles in eukaryotic cells.

[0133] In some cases, at least one nonviral RNA molecule contains one aptamer sequence. In other cases, at least one nonviral RNA molecule may contain aptamer sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. For example, in some cases, a lentivirus-like particle contains at least one nonviral RNA molecule containing two aptamer sequences. If a lentivirus-like particle contains CRISPR-associated endonuclease mRNA and gRNA, either or both may contain one or more aptamer sequences. In one example, if a particle contains both CRISPR-associated endonuclease mRNA and gRNA, the CRISPR-associated endonuclease mRNA may contain one or more aptamer sequences located at its 3' end. In another example, if a particle contains both CRISPR-associated endonuclease mRNA and gRNA, the gRNA may contain one or more aptamer sequences. In another example, if a particle contains both CRISPR-related endonuclease mRNA and gRNA, both the CRISPR-related endonuclease mRNA and gRNA may contain one or more aptamer sequences. If a particle contains both CRISPR-related endonuclease mRNA and gRNA, and both contain one or more aptamer sequences, the CRISPR-related endonuclease mRNA and gRNA may contain the same aptamer sequence or different aptamers.

[0134] In some cases, the lentivirus-like particle contains a gRNA containing a CRISPR-associated endonuclease mRNA having at least one first aptamer sequence located at its 3' end, and at least one second aptamer sequence. In one example, the at least one first aptamer sequence and the at least one second aptamer sequence are identical aptamer sequences. In some cases, the at least one first aptamer sequence is an aptamer sequence to which a first ABP specifically binds, and the at least one second aptamer sequence is an aptamer sequence to which a second ABP specifically binds, and the at least one first aptamer sequence and the at least one second aptamer sequence bind different first and second ABPs. For example, the at least one first aptamer sequence and the at least one second aptamer sequence may specifically bind an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA-binding domain, and COM protein. In another example, at least one first aptamer sequence and at least one second aptamer sequence may each be specifically bound to different ABPs selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA-binding domain, and COM protein.

[0135] In some cases, nonviral RNA molecules may also contain RNA stabilization sequences located at the 3' end of CRISPR-associated endonuclease mRNA, or, if at least one aptamer sequence is located at the 3' end of the nonviral RNA molecule, at least one aptamer sequence located after it. The RNA stabilization sequences stabilize the longevity of the nonviral RNA molecule. In one example, a nonviral RNA molecule contains CRISPR-associated endonuclease mRNA with one or more aptamer sequences and an RNA stabilization sequence after one or more aptamer sequences. In another example, a nonviral RNA molecule contains gRNA and an RNA stabilization sequence downstream of the 3' end of the gRNA (after any aptamer sequence located at the 3' end of the gRNA). An exemplary RNA stabilization sequence is the sequence of the 3'UTR of the human betaglobin gene, as shown in SEQ ID NO: 25 (DNA) and SEQ ID NO: 26 (RNA). Other RNA stabilization sequences are described in Hayashi, T. et al., Developmental Dynamics 239(7):2034-2040 (2010) and Newbury, S. et al., Cell 48(2):297-310 (1987).

[0136] A characteristic of the provided lentivirus-like particles is the amount of nonviral RNA molecules that can be packaged within the particle. In natural lentiviruses, the viral shell (capsid) contains two copies of a single-stranded RNA genome. For example, a wild-type HIV-1 virus particle contains two copies of a single-stranded RNA genome of approximately 9.7 kb. In the lentivirus-like particles of this disclosure, up to 100 copies of nonviral RNA molecules can be packaged within the viral shell. For example, a lentivirus-like particle may contain approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nonviral RNA molecules. For example, a particle may contain 5-25 nonviral RNA molecules, 25-50 nonviral RNA molecules, 50-75 nonviral RNA molecules, or 75-100 nonviral RNA molecules. For example, a particle may contain approximately 50 to 100 nonviral RNA molecules. In some cases, lentivirus-like particles may contain up to 100 copies of CRISPR-associated endonuclease mRNA (e.g., SaCas9 mRNA). In some cases, lentivirus-like particles may contain several copies of CRISPR-associated endonuclease mRNA, gRNA, or a combination thereof. In some cases, lentivirus-like particles may contain several copies of nonviral RNA molecules containing at least one aptamer sequence. In other cases, lentivirus-like particles may contain several copies of nonviral RNA molecules that do not contain at least one aptamer sequence. In some cases, an aptamer sequence is not required to produce lentivirus particles containing several copies of nonviral RNA molecules.

[0137] In some cases, lentivirus-like particles contain RNPs, which are complexes of CRISPR-associated endonucleases and gRNAs. In some cases, lentivirus-like particles comprise a) a fusion protein containing a nucleocapsid (NC) protein or a matrix (MA) protein, wherein the NC protein or MA protein contains at least one nonviral aptamer-binding protein (ABP), and b) a ribonucleotide protein (RNP) complex containing a CRISPR-associated endonuclease and guide RNA. In some cases, lentivirus-like particles comprise a fusion protein containing a viral protein R (VPR) protein or a negative regulator (NEF) protein, wherein the VPR protein or NEF protein contains at least one nonviral aptamer-binding protein (ABP), and b) a ribonucleotide protein (RNP) complex containing a CRISPR-associated endonuclease and guide RNA. In lentivirus-like particles containing RNPs, the guide RNA contains an aptamer that binds to the nonviral aptamer-binding protein of the fusion protein.

[0138] Lentivirus-like particles containing RNP can be constructed using any of the mammalian expression plasmids described herein, which include a nonviral nucleic acid sequence encoding a CRISPR-related endonuclease and a gRNA sequence, and in which at least one aptamer is attached to or inserted into the gRNA sequence. As described above, the interaction between the aptamer sequence attached to or inserted into the gRNA in the viral fusion protein described herein and the aptamer-binding protein facilitates the packaging of the gRNA, which is complexed with CRISPR-related endonuclease (RNP), into the lentivirus particle. VI.Cells

[0139] Furthermore, cells containing the compositions described herein and cells modified by the compositions described herein are also provided. Cells or populations of cells containing one or more nucleic acid constructs (plasmids) described herein are also provided. For example, cells containing the lentiviral packaging plasmid described above are provided herein. For example, cells containing the mammalian expression plasmid described above are provided herein. In another example, cells containing both the lentiviral packaging plasmid and the mammalian expression plasmid described above are provided herein. In this method, lentiviral-like particles containing the CRISPR components of the system described herein can be produced. In another example, cells containing the lentiviral-like particles described above are provided herein. In this method, modification of a target DNA sequence in the cell genome can be achieved by the CRISPR components of the system described herein. Cells or populations of cells containing the lentiviral packaging system described herein are also provided. Cells or populations of cells containing the lentiviral-like particles described herein are also provided.

[0140] Cells include, but are not limited to, eukaryotic cells, prokaryotic cells, human cells, non-human animal cells, and fungal cells. If necessary, the cells are in a cell culture. If necessary, the cells are mammalian cells, e.g., human cells. The cells may be in vitro, ex vivo, or in vivo. The cells may also be primary cells, embryonic cells, stem cells, or precursor cells. Precursor cells may be, for example, pluripotent stem cells or hematopoietic stem cells. The introduction of the composition into cells may be cell cycle-dependent or cell cycle-independent. Methods for synchronizing cells to increase the proportion of cells in a particular phase are known in the art. Depending on the type of cells to be modified, those skilled in the art can easily determine whether cell cycle synchronization is necessary. In some examples, cells are harvested from a subject, modified using one of the methods described herein, and administered to the subject.

[0141] As needed, the cell is a T cell. T cells include, but are not limited to, untreated T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof or small populations thereof. T cells are CD4 + , CD8 + , or CD4 + and CD8 + and can be. T cells are helper cells, e.g., T H 1, T H 2, T H 3, T H 9, T H 17 or T FH type helper cells and can be. T cells can be cytotoxic T cells. T cells can be recombinant T cells that have been genetically engineered, e.g., T cells that express a chimeric antigen receptor or a recombinant T cell receptor. VII. Gene Editing Methods

[0142] Methods of using the plasmids and systems provided in this disclosure in the CRISPR / Cas system to edit a DNA target or modulate the transcription of one or more DNA targets are described herein. These methods can be used to suppress, mutate or activate a target genomic sequence, e.g., a gene, in the genome of a eukaryotic cell.

[0143] In the method provided herein, eukaryotic cells containing the target genome sequence to be modified are transduced using lentivirus-like particles containing a viral fusion protein comprising a viral protein fused to at least one aptamer-binding protein (ABP) and CRISPR-related endonuclease mRNA. A characteristic feature of the described method is that the eukaryotic cells do not stably express CRISPR-related endonuclease. CRISPR-related endonuclease is not constitutively expressed; rather, a finite amount of CRISPR-related endonuclease mRNA is provided to the transduced cells via the lentivirus-like particles. The advantage of the provided method is a reduced off-target gene editing event because CRISPR-related endonuclease is not present in the cells and, as such, is inactive for a long period of time. Additionally, when lentivirus-like particles lacking integrase activity are used in the method, there is a reduced risk of integration of any of the nucleic acids held by the particles, particularly the CRISPR-related endonuclease coding sequence, into the cellular genome. In some cases, the lentiviral particles used lack a portion of the lentiviral genome sequence essential for viral replication, thereby reducing the risk of continued particle production. Another advantage of the provided components is that the viral fusion protein can increase the packaging of nonviral RNA molecules, such as CRISPR-associated endonuclease mRNA, into lentiviral-like particles, which enhances genome editing efficiency. In some cases, the nonviral RNA molecules packaged into lentiviral-like particles contain at least one aptamer sequence located at the 3' end.

[0144] In some cases, transduced eukaryotic cells are mammalian cells. In some cases, eukaryotic cells may be in vitro cultured cells. In some cases, eukaryotic cells may be ex vivo cells obtained from the subject. In other cases, eukaryotic cells are present in the subject. As used throughout, subject means individual. For example, subject is a mammal, e.g., primate, more specifically, human. Non-human primates are also subjects. The term subject includes domesticated animals, e.g., cats, dogs, livestock (e.g., cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Veterinary and medical uses and formulations are thus intended herein. This term does not indicate a specific age or sex. Therefore, adult and neonatal subjects, whether male or female, are considered subjects. As used herein, patient or subject may be used synonymously and may refer to a subject suffering from a disease or disorder. The viral particles of the system provided herein may be injected into a subject according to known, routine methods. In some cases, the viral particles of the system may be injected intravenously (IV), intraperitoneally (IP), intramuscularly, or into a specific organ.

[0145] In some cases, the methods provided are for modifying a target gene locus of interest, and the methods involve transducing multiple eukaryotic cells using multiple viral particles, the multiple viral particles being fusion proteins comprising a viral protein R (VPR) protein or a negative regulator (NEF) protein, the VPR protein or NEF protein comprising at least one nonviral aptamer-binding protein (ABP), and a ribonucleotide protein (RNP) complex comprising a CRISPR-associated endonuclease and guide RNA, the RNP binding to a genomic target sequence in the cell's genomic DNA, the CRISPR-associated endonuclease cleaving the cell's genomic DNA, thereby inducing a cellular DNA repair mechanism and causing modification of the genomic target sequence. In some cases, multiple viral particles comprise a) a fusion protein containing a nucleocapsid (NC) protein or a matrix (MA) protein, wherein the NC protein or MA protein contains at least one nonviral aptamer-binding protein (ABP), and b) a ribonucleotide protein (RNP) complex containing a CRISPR-associated endonuclease and guide RNA, wherein the RNP binds to a genomic target sequence in the cell's genomic DNA, and the CRISPR-associated endonuclease cleaves the cell's genomic DNA, thereby inducing a cellular DNA repair mechanism and causing modification of the genomic target sequence.

[0146] As described above, RNPs are packaged within viral particles by attachment to a gRNA sequence that binds to at least one nonviral aptamer-binding protein in the fusion protein described herein, or by the interaction of an aptamer sequence inserted into the gRNA sequence. The gRNA sequence binds to at least one nonviral aptamer-binding protein in the fusion protein described herein and interacts with a CRISPR-associated endonuclease to form a complex (RNP).

[0147] In some cases, the methods provided are for modifying a target gene locus of interest, and the methods include delivering a CRISPR-related endonuclease and one or more nucleic acid components to the locus, the CRISPR-related endonuclease forming a complex with the one or more nucleic acid components, and upon binding of the complex to the target gene locus, the CRISPR-related endonuclease induces modification of the target gene locus of interest. In preferred embodiments, the modification is the introduction of strand breaks. In connection with this disclosure, lentivirus-like particles containing CRISPR-related endonuclease mRNA are used to transduce cells containing a target gene locus of interest to be modified, thereby expressing CRISPR-related endonuclease in the cells from the CRISPR-related endonuclease mRNA.

[0148] The provided method allows genome editing to be achieved using lentivirus-like particles containing CRISPR-associated endonuclease mRNA, together with other viral particles providing gRNA components for the editing system. In some cases, multiple eukaryotic cells are transduced using lentivirus-like particles containing both CRISPR-associated endonuclease mRNA and gRNA. In some cases, multiple eukaryotic cells are transduced using lentivirus-like particles containing CRISPR-associated endonuclease mRNA and a second viral particle providing gRNA or a gRNA-coding sequence. If the second viral particle is a type of virus containing an RNA genome, the viral particle may contain gRNA or a gRNA-coding sequence. For example, the second viral particle may be a lentivirus-like particle containing gRNA. In another example, the second viral particle may be a lentivirus particle containing a gRNA-coding sequence. If the second viral particle is a type of virus containing a DNA genome, the viral particle contains a gRNA-coding sequence in which the gRNA-coding sequence is operably linked to a eukaryotic cell promoter. In another example, the second viral particle may be an adenovirus or adeno-associated virus containing a gRNA coding sequence operably linked to a eukaryotic cell promoter.

[0149] In some cases, the methods provided for genome editing or modifying a sequence associated with or located at a target locus of interest include introducing CRISPR / Cas system components, including CRISPR-associated endonuclease mRNA, into a eukaryotic cell, thereby enabling the CRISPR / Cas system components to function efficiently to integrate the DNA insertion portion into the eukaryotic cell's genome. In certain embodiments, the genome is a mammalian genome. In some cases, the integration of the DNA insertion portion is facilitated by non-homologous end joining (NHEJ) or homology-directed recombination (HDR). In some cases, the DNA insertion portion is an exogenously introduced target template sequence, e.g., a DNA template or repair template. The target template sequence includes a nucleic acid sequence having the desired insertion or modification into a genomic target sequence present in the eukaryotic cell. The target template sequence also includes nucleic acid sequences homologous to genomic DNA adjacent to the genomic target sequence.

[0150] The target template sequence can be introduced into eukaryotic cells by transduction using adenovirus particles, adeno-associated viruses, or embedded defective lentivirus particles containing the target template sequence. In some cases, the adenovirus particles, adeno-associated viruses, or embedded defective lentivirus particles may also contain a gRNA coding sequence operably ligated to a eukaryotic cell promoter.

[0151] In homologous recombination repair, the target template sequence acts as a donor template, and the cell's innate DNA repair mechanism results in the insertion of a nucleic acid sequence with the desired insertion or modification into the genomic target sequence. Genomic modifications performed in this manner can be used to insert novel genes or to knock out existing genes. NHEJ-mediated repair of CRISPR / Cas system breaks tends to produce indel errors, and may therefore be useful when the intention is to create a null allele ("knockout") in the target genomic sequence. Indel errors that occur during the process of repair by NHEJ are typically small (1–10 bp) but highly heterogeneous. As a result, there is an opportunity for about two-thirds of NHEJ-mediated repairs to result in frameshift mutations. NHEJ does not obligately introduce indels. Considering the terminal structure of double-strand breaks (blunt or nearly blunt ends without nucleotide damage), indels are rare and, in some cases, account for less than 5% of repair events. However, while the products of accurate repair are readily re-cleaved, indel products are not (they no longer pair with gRNA), and therefore, prolonged exposure of genomic DNA to CRISPR system components is favorable for the accumulation of the latter products. In some cases, pairs of gRNAs adjacent to hundreds of base pairs or more can simultaneously introduce pairs of chromosomal breaks when the NHEJ joins their distal ends together, resulting in the deletion of intervening DNA ("pop-out" deletion). Similarly, provided that a target template sequence containing a suitable overhang is used, it may be possible to directly insert exogenous DNA fragments via NHEJ-dependent repair ("pop-in" insertion) at CRISPR-associated endonuclease-targeted breaks (or pairs of breaks).

[0152] The methods described may be used with any CRISPR-related endonuclease that requires a constant region of sgRNA for its function. These include, but are not limited to, RNA-induced site-specific nucleases. Examples include nucleases present in any bacterial species encoding a type II or type V CRISPR / Cas system. Suitable CRISPR-related endonucleases are described throughout this disclosure. For example, but are not limited, site-specific nucleases may be Cas9 polypeptides or Cpf1 polypeptides, or derivatives thereof.

[0153] In some cases, the CRISPR-associated endonuclease may be an active Cas9 polypeptide, and if it is bound to the target nucleic acid as part of a complex with gRNA or as part of a complex with a DNA template, a double-strand break is induced in the target nucleic acid. The double-strand break may be repaired by an NHEJ to introduce a random mutation, or by an HDR to introduce a specific mutation. Various Cas9 nucleases can be used in the methods described herein. For example, a Cas9 nuclease that requires an NGG protospacer adjacent motif (PAM) immediately 3' of the region targeted by the guide RNA can be used. Such a Cas9 nuclease can target any region of the genome containing an NGG sequence. As another example, a Cas9 protein with an orthogonal PAM motif requirement can be used to target sequences that do not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, the Cpf1 protein and its fragments and derivatives, as described in Esvelt et al., Nature Methods 10 (11):1116-1121 (2013), and those described in Zetsche et al., Cell 163 (3):759-771 (2015).

[0154] In some cases, the CRISPR-associated endonuclease is a modified Cas9 protein that is a nickase, and as a result, when bound to the target nucleic acid as part of a complex with gRNA, a single-strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally different guide RNA, can target two proximal sites in the target genomic region and thus introduce a pair of proximal single-strand breaks into the target genomic region. Since off-target effects are generally likely to result in a single nick that is repaired without damage by the base excision repair mechanism, the nickase pair can provide enhanced specificity. Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations.

[0155] For example, if a lentivirus-like particle encodes a Cas9 polypeptide that generates blunt-ended double-strand breaks, the method can be used to render the gene incapacitated, as repair can proceed readily by NHEJ. In another example, if a lentivirus-like particle encodes a Cpf1 polypeptide that generates sticky ends, the method can be useful in incorporating a novel sequence of DNA, such as for gene insertion or to create a knock-in. In some cases, the Cpf1 protein can be used to bring about gene transfer. However, any CRISPR-related endonuclease can be used to introduce a gene sequence or remove (incapacitate) a gene. In some cases, a modified Cas9 nickase can be used to introduce single-strand breaks close together on opposite strands, resulting in a DSB with a long overhang.

[0156] Generally, sgRNAs are targeted to specific regions of a gene or its vicinity. In some cases, sgRNAs are targeted to regions that affect gene transcription. For example, sgRNAs can be targeted to a region of 0–750 bp 5' (upstream) of a gene's transcription start site, or to a region in its vicinity. In some cases, targeting of the 0–750 bp region can provide, or may provide, increased transcriptional activation by the sgRNA:inactivated CRISPR-related endonuclease. For example, sgRNAs can form a complex with an inactivated CRISPR-related endonuclease, such as a dCas9 polypeptide to a transcription activator, thereby providing increased transcriptional activation of the gene by the complex. As another example, sgRNAs can be targeted to a region of 0–1000 bp 3' (downstream) of a gene's transcription start site, or to a region in its vicinity. In some cases, targeting this region may be done to provide increased transcriptional repression by the sgRNA:inactivated CRISPR-related endonuclease complex. For example, sgRNA can form a complex with a CRISPR-related endonuclease, such as a dCas9 polypeptide linked to a transcription inhibitor, thereby providing increased transcriptional repression of the gene by the complex.

[0157] In some cases, sgRNAs are targeted to genomic regions that are predicted to contain relatively few nucleosomes. Nucleosome localization and occupancy can be assayed using enzymatic digestion with micrococcal nucleases (MNases), for example, by MNase-seq analysis. Therefore, in some cases, sgRNAs are targeted to genomic regions with low MNase-seq signals. In some cases, sgRNAs are targeted to regions that are predicted to be highly transcriptionally active. For example, sgRNAs may be targeted to regions that are predicted to have relatively high occupancy for RNA polymerase II (Pol II). Such regions can be identified by Pol II chromatin immunoprecipitation sequencing (ChIP-seq). Therefore, in some cases, sgRNAs are targeted to regions with high Pol II ChIP-seq signals, such as those disclosed in the ENCODE-published Pol II ChIP-seq database (Landt, et al., Genome Research 22(9):1813-1831 (2012)). In another example, sgRNAs are targeted to run-on sequencing or global run-on sequencing. As identified by GRO-seq sequencing, regions predicted to be highly transcriptionally active can be targeted. Therefore, in some cases, sgRNAs can be targeted to publicly available GRO-seq data (e.g., Core et al., Science. 2008 Dec 19;322(5909):1845-8; and Hah et al. It targets regions with high GRO-seq signals as disclosed in al., Genome Res. 2013 Aug;23(8):1210-23).

[0158] In some cases, modifications to system components as described in this disclosure do not impair how the system components function after transduction into eukaryotic cells. Rather, the components may function as well as, or better than, the unmodified components during transduction into eukaryotic cells. For example, viral fusion proteins in lentivirus-like particles may not interfere with the transduction of eukaryotic cells into lentivirus-like particles. Similarly, if a nonviral RNA molecule packaged in a lentivirus-like particle contains at least one aptamer sequence, this at least one aptamer sequence may not interfere with the transduction of eukaryotic cells into lentivirus-like particles. For example, if the nonviral RNA molecule is a CRISPR-related endonuclease mRNA, the presence of at least one aptamer sequence may not impair the expression of the CRISPR-related endonuclease mRNA molecule. In another example, a CRISPR-associated endonuclease expressed from CRISPR-associated endonuclease mRNA, along with at least one aptamer sequence immediately downstream, may be as functional as one expressed from an mRNA molecule lacking that at least one aptamer sequence. In yet another example, a gRNA containing at least one aptamer sequence may be as functional as a gRNA lacking any aptamer sequence. In some cases, lentivirus-like proteins containing a viral fusion protein may result in greater gene editing upon transduction into eukaryotic cells compared to lentivirus-like particles without a viral fusion protein. In one example, the viral fusion protein may be an NC-ABP fusion protein, e.g., an NC-MS2 fusion protein or an NC-PP7 fusion protein. In one example, the NC fusion protein is fused to one or two ABPs, e.g., one or two MS2 proteins, one or two PP7 proteins, or one MS2 protein and one PP7 protein.

[0159] Eukaryotic cells may be in vitro, ex vivo, or in vivo. In some embodiments, the cells are primary cells (isolated from the subject). As used herein, primary cells are cells that have not been transformed or immortalized. Such primary cells may be cultured, subcultured, or passaged a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some examples, primary cells are adapted to in vitro culture conditions. In some examples, primary cells are isolated from an organism, system, organ, or tissue, sorted as needed, and utilized directly without culture or subculture. In some examples, primary cells are stimulated, activated, or differentiated. In some embodiments, cells are cultured under conditions effective for expanding a population of modified cells. In some embodiments, cells modified by any of the methods provided herein are purified. In some cases, cells are harvested from the subject, modified using one of the methods described herein, and re-administered to the patient.

[0160] In some cases, once cells have been transduced using the aforementioned viral particles, they are cultured for a sufficient amount of time for gene editing to occur, and as a result, a pool of cells expressing a detectable phenotype can be selected from multiple transduced cells. The phenotype may be, for example, cell growth, survival, or proliferation. In some cases, the phenotype is cell growth, survival, or proliferation in the presence of drugs, e.g., cytotoxic agents, oncogenes, tumor suppressors, transcription factors, kinases (e.g., receptor tyrosine kinases), genes under the control of promoters (e.g., xenopromoters) (e.g., exogenous genes), checkpoint genes or cell cycle regulators, growth factors, hormones, DNA damaging agents, drugs, or chemotherapeutic agents. The phenotype may also be protein expression, RNA expression, protein activity, or cell motility, migration, or invasion. In some cases, cell selection based on phenotype includes cell fluorescence activation, affinity purification, or selection based on cell motility.

[0161] In some cases, cell selection includes, for example, analysis of the cell's genomic DNA by amplification, sequencing, and SNP analysis. Sequencing methods include, but are not limited to, shotgun sequencing, bridge PCR, Sanger sequencing (including microfluidic Sanger sequencing), pyrosequencing, massively parallel signature sequencing, nanopore DNA sequencing, single-molecule real-time sequencing (SMRT) (Pacific Biosciences, Menlo Park, California), ion semiconductor sequencing, ligation sequencing, synthetic sequencing (Illumina, San Diego, California), Polony sequencing, 454 sequencing, solid-phase sequencing, DNA nanoball sequencing, heliscope single-molecule sequencing, mass spectroscopy sequencing, pyrosequencing, Supported Oligo Ligation Detection (SOLiD) sequencing, DNA microarray sequencing, RNAP sequencing, tunnel current DNA sequencing, and any other DNA sequencing methods to be identified in the future. One or more of the sequencing methods described herein can be used in high-throughput sequencing methods. As used herein, the term “high-throughput sequencing” refers to all methods relating to sequencing nucleic acids in which more than one nucleic acid sequence is sequenced in a given time.

[0162] Other studies have evaluated the usefulness of using lentiviral particles to deliver CRISPR / Cas components to eukaryotic cells. Choi et al., Gene Therapy 23(7): 627-633 (2016) described a system in which a Cas9 coding sequence was inserted into a lentiviral genome, resulting in lentiviral particles containing the Cas9 protein. This system has low viral titer and low gene editing activity. Compared to Choi et al.'s system, the advantages of the system and components described herein include: 1) production of lentiviral-like particles with titers similar to conventional lentiviral systems; 2) packaging of multiple CRISPR-related endonuclease mRNA molecules into each lentiviral-like particle, with each mRNA useful for translation of multiple CRISPR-related endonuclease proteins; and 3) the CRISPR-related endonucleases expressed from the mRNA molecules packaged into the lentiviral-like particles are sufficiently active and do not require post-translational processing. For example, Choi et al. describe obtaining an indel rate of 30% (using particles reflecting 150 ng of P24). In contrast, when using the lentivirus-like particles described herein, the inventors of this disclosure observed an indel rate of over 80% (45 ng of P24; 2 × 10⁻¹⁶). 4 Individual HEK293T cells; particles reflecting sickle cell mutation correction.

[0163] Other studies have evaluated the usefulness of using lentiviral particles to deliver mRNA to eukaryotic cells (Mock et al., Scientific Reports 4: 6409 (2014) and Prel et al., Molecular Therapy Methods & Clinical Development). 2: 15039 (2015)). None of the groups evaluated the effectiveness of using lentivirus-like particles to introduce CRISPR / Cas system components into eukaryotic cells. Mock et al. delivered TALEN mRNA molecules using a lentiviral genome. In this system, only two copies of TALEN mRNA molecules were packaged per viral particle. The degree of translation from TALEN mRNA was relatively low, leading to insufficient gene editing activity. The inventors of this disclosure tested Mock et al.'s system to deliver SaCas9 mRNA molecules to GFP reporter eukaryotic cells useful for detecting gene editing events. Lentiviruses with a SaCas9 coding sequence inserted into their genome were packaged using a packaging plasmid encoding inactivated reverse transcriptase. The resulting lentiviral particles were co-transduced into GFP reporter cells as described in the examples, together with a lentivirus expressing HBB sgRNA1. The GFP+ reporter cells were undetectable, indicating that no gene editing occurred. Thus, the system described by Mock et al. was not useful for CRISPR / Cas system gene editing. Prel et al. delivered Cre mRNA molecules into eukaryotic cells using lentiviral particles in vivo and in vitro. The lentiviral-like particles contained an NC fusion protein in which the second zinc finger domain of the NC protein was replaced with an MS2 protein. The Cre mRNA also had 12 copies of the MS2 aptamer sequence attached to the 3'UTR. Their system had inefficient viral particle production and low copy number packaging of Cre mRNA molecules into viral particles. In comparison to Prel et al.'s system, the system of this disclosure efficiently generates viral particles and has a high copy number of nonviral RNA molecules per viral particle. Prel et al. reported observing packaging of 5–6 copies of Cre mRNA per particle.In contrast, using the lentivirus-like particles described in this disclosure (plasmid number 11 as a packaging plasmid) prepared to express the NC-MS2 fusion protein, the inventors of this disclosure observed 100 copies of SaCas9 mRNA per particle when the SaCas9 mRNA was modified with human HBB 3'UTR (plasmid number 36), or 30 copies of SaCas9 mRNA per particle when human HBB 3'UTR was not added (plasmid number 16). VIII. Treatment Methods

[0164] Any of the methods and compositions described herein can be used to treat diseases in a subject (e.g., cancer, blood disorders (e.g., sickle cell anemia or beta-thalassemia), infectious diseases, autoimmune diseases, transplant rejection, graft-versus-host disease, or other inflammatory disorders).

[0165] In some methods, the cancers to be treated are selected from B-cell origin cancers, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, colon cancer, chronic myeloid cancer, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia (CLL), or acute lymphocytic leukemia (ALL)), prostate cancer, colon cancer, renal cell carcinoma, liver cancer, kidney cancer, ovarian cancer, stomach cancer, testicular cancer, rhabdomyosarcoma, and Hodgkin lymphoma. In some embodiments, B-cell origin cancers are selected from the group consisting of lineage B acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin lymphoma.

[0166] In some methods, the cells of interest are modified in vivo. In some methods, a method for treating a disease in a subject comprises a) obtaining cells from the subject, b) modifying the cells using one of the methods provided herein, and c) administering the modified cells to the subject. Where necessary, the disease is selected from the group consisting of cancer, hematological disorders (e.g., sickle cell anemia or beta-thalassemia), infectious diseases, autoimmune diseases, transplant rejection, graft-versus-host disease, or other inflammatory disorders in the subject. In some methods for treating cancer, cells obtained from the subject are modified to express tumor-specific antigens. As used throughout, the term “tumor-specific antigen” means an antigen that is unique to cancer cells or expressed in greater quantities in cancer cells than in non-cancerous cells. Where necessary, the cells obtained from the subject are T cells. Where necessary, the modified cells are enlarged before administration to the subject.

[0167] All patents, patent publications, patent applications, journal articles, books, technical references, etc., discussed herein are incorporated herein by reference in their entirety for all purposes.

[0168] It should be understood that the drawings and descriptions in this disclosure are simplified to illustrate elements relevant to a clear understanding of this disclosure. Naturally, the drawings are presented for illustrative purposes only, not as structural diagrams. Details omitted and modifications or alternative embodiments are within the scope of those skilled in the art.

[0169] In certain aspects of this disclosure, it should be understood that a single component may be replaced by multiple components, and multiple components may be replaced by a single component, in order to provide an element or structure or to perform a given function (one or more). Such substitutions are intended to be within the scope of this disclosure unless they do not function to carry out a particular embodiment of this disclosure.

[0170] The examples presented herein are intended to illustrate potential and specific implementations of the Disclosure. It can be understood that the examples are intended primarily to illustrate the Disclosure to those skilled in the art. Variations of these drawings or operations described herein are possible without departing from the spirit of the Disclosure. For example, in certain particular cases, method steps or operations may be performed or carried out in a different order, and operations may be added, omitted, or modified.

[0171] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intervening value between the upper and lower limits of that range, down to the smallest unit of the lower limit, is also specifically disclosed. Any narrower range between any listed or unlisted intervening value within the stated range and any other listed or intervening value within that stated range is included. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which either or both limits are included in or excluded from that smaller range is also included within the art affected by any specifically excluded limits within the stated range. Where the stated range includes one or both limits, it also includes ranges that exclude either or both of those included limits.

[0172] There may be different arrangements of components and steps shown in the drawings or described above, as well as components and steps not shown or described. Similarly, some characteristics and partial combinations may be useful and may be used without reference to other characteristics and partial combinations. The embodiments described herein are for illustrative purposes only and not limiting purposes, but alternative embodiments will be apparent to the reader of this patent. Thus, this disclosure is not limited to the embodiments described above or shown in the drawings, and various embodiments and modifications can be made without departing from the scope of the following claims. [Examples]

[0173] (Example 1) material and method Next-generation sequencing and data analysis. Genomic DNA was isolated from cells using the QIAamp DNA Mini kit (Qiagen, Germantown, Maryland) according to the manufacturer's instructions. Two DNA regions were amplified for next-generation sequencing analysis. A nested PCR strategy was used to amplify the endogenous HBB target sequence for sequencing, avoiding amplification of sequences derived from the viral vector template. First, a 3.4 kb region from the HBB locus was amplified using primers HBB-1849F and HBB-5277R (SEQ ID NOs. 96 and 97). These two primers cannot amplify sequences from the template in the viral vector. Next, the target DNA was amplified for sequencing using HBB-MUT-F and HBB-MUT-R primers (Table 3; SEQ ID NOs. 34-40). For sequencing, reporter-mut-F and reporter-mut-R primers were used to amplify the HBB target site from the embedded EGFP reporter (Table 3; SEQ ID NOs. 98-102). For PCR, proofreading HotStart® ReadyMix from KAPA Biosystems (Wilmington, Massachusetts) was used. PCR products were analyzed by next-generation sequencing using Illumina NextSeq 500, as previously described by Javidi-Parsijani, P. et al., PLoS One 2017; 12(5): e0177444. For mutation analysis of the sequencing data, Cas-Analyzer online software, as described by Park, J. et al., Bioinformatics 33(2):286-288 (2017), was used.

[0174] Plasmids: pRSV-Rev (Addgene catalog number 12253), pMD2.G (Addgene catalog number 12259), pMDLg / pRRE (Addgene catalog number 12251), psPAX2 (Addgene catalog number 12260), psPAX2-D64V (Addgene catalog number 63586), pSL-MS2x12 (Addgene catalog number 27119), and pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (Addgene catalog number 61591). pCDH-GFP was purchased from Systems Biosciences Inc. (catalog number CD513B-1). The plasmids listed in Table 2 were manufactured by the inventors of this disclosure and used in the following examples. Synthetic DNA sequences, such as those identified below, were synthesized by GenScript on special order. The primers listed in Table 3 below were synthesized by Eurofins Genomics on a special order basis. Table 2. Plasmids used in the examples [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] Table 3. Primers used in the examples [Table 3-1] [Table 3-2]

[0175] An assay for evaluating gene editing activity. To evaluate gene editing, a mutated sequence that causes sickle cell disease was selected as the genomic target sequence for editing. The gene defect that causes sickle cell disease is a point mutation in the human beta-hemoglobin gene, where the A nucleotide is replaced by a T nucleotide (converting the GAG ​​codon to GUG). As a result, glutamic acid (E / Glu) at amino acid position 6 of the β-globin protein is replaced by valine (V / Val). The sequence of the targeted chain is: [ka] The first six nucleotides of this sequence (underlined) correspond to the protospacer-adjacent motif. Gene editing activity was detected using two assays: (1) a GFP-reporter assay and (2) the Surveyor® Mutation Detection Kit (Integrated DNA Technologies, catalog number 706020).

[0176] For the GFP-reporter assay, enhanced green fluorescent protein (EGFP) reporter cells (referred to as "GFP reporter cells" or "reporter cells" throughout the examples) described in Javidi-Parsijani, P. et al., PLoS One 2017; 12(5): e0177444 were used for detection of genome editing activity by microscopic observation or flow cytometry analysis of GFP-positive cells. This cell line does not express EGFP by disrupting the EGFP reading frame by inserting a 119 base pair sequence between the start codon ATG and the second codon of the EGFP cDNA (the insertion loses 1 bp to create 40 in-frame codons). The 119 bp insertion is [ka] It contains a 57 bp IL2RG target sequence (underlined) and a 62 bp HBB target sequence (capitalized). Without genome editing, EGFP is not expressed due to the presence of an insertion that disrupts the EGFP reading frame. This was confirmed by the absence of EGFP-positive cells in untransfected reporter cells and in reporter cells transfected with plasmid DNA expressing SaCas9 without any sgRNA. During genome editing at the inserted sequence, double-strand breaks are repaired by non-homologous end joining. By introducing an insertion or deletion (indel), a 3N+2 base pair deletion or a 3N+1 base pair insertion restores the EGFP reading frame and, therefore, EGFP protein expression.

[0177] To test whether the reporter cells were functional, cells were transduced using adeno-associated virus particles (pSaCas9-HBB-sgRNA1; produced from plasmid number 22) containing SaCas9 mRNA and HBB sgRNA. Flow cytometry analysis showed that 4.5%–15% of the cells were EGFP-positive 24 hours after transfection. Genomic DNA obtained from reporter cells 3 days after transfection was purified, the reporter region was amplified by PCR using high-fidelity DNA polymerase, and the target region was sequenced by next-generation sequencing. The percentage of sequences containing indels was analyzed. 68% of the reads were found to contain indels. Since only some indels restore EGFP expression, it is not surprising that the EGFP positivity rate was lower than the indel rate when the analysis was performed 72 hours after transfection. These data indicate that the reporter cells are functional and useful for analyzing gene editing events in relation to this disclosure.

[0178] Gene editing activity was detected using the Surveyor® Mutation Detection Kit according to the kit's manual. A key component of the kit, Surveyor nuclease, a member of the CEL family of mismatch-specific nucleases, recognizes and cleaves mismatches resulting from the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. Briefly, the target sequence was amplified using proofreading HotStart ReadyMix, a high-fidelity, thermostable DNA polymerase from KAPA Biosystems (Wilmington, Massachusetts), to minimize the incorporation of errors resulting in background mismatches. The amplified DNA was denatured at 95°C for 5 minutes and then regenerated by slowly decreasing the temperature to room temperature. The DNA was then treated with Surveyor nuclease to cleave mismatches. The cleaved DNA fragments were separated by agarose gel electrophoresis, stained with ethidium bromide, and observed under UV light.

[0179] Packaging of Cas9 mRNA into lentivirus-like particles. Lentivirus-like particles containing the genome were produced using the Addgene second or third-generation packaging system as described in Javidi-Parsijani, P. et al., PLoS One 2017; 12(5): e0177444. To package SaCas9 mRNA into lentivirus-like particles, desired combinations of plasmids were transfected into HEK293T cells. Transfection was mediated by polyethyleneimine (PEI, Polysciences Inc.) using a 1:2 DNA:PEI ratio (mass:mass). Table 4 provides exemplary conditions used to produce lentivirus-like particles with a particular Cas9 mRNA packaged. Cell culture and DNA transfection were described in Javidi-Parsijani, P. et al., PLoS One 2017; 12(5): The procedure was carried out as described in e0177444. Virus particles were used either without concentration or after concentration using one of three different methods: 1) by layering the supernatant containing virus particles onto a 10 ml 20% sucrose cushion and then centrifuging at 20,000 g, 4°C for 4 hours; 2) by using a Lenti-X® concentrator according to the manufacturer's protocol (Clontech, catalog no. 631232); or 3) after concentration using a KR2i TFF system [KrosFlo® Research 2i Tangential Flow Filtration system] (Spectrum Lab, catalog no. SYR2-U20). Transduction experiments showed that the virus produced by the third method worked best (determined by the percentage of GFP-positive cells generated in the GFP reporter assay), and therefore, most of the data was generated using virus particles concentrated by this method. Table 4. Plasmid DNA used to transfect HEK293T cells to produce lentiviral particles loaded with SaCas9 mRNA. a [Table 4-1] [Table 4-2] a 13 x 10 24 hours before transfection 6 The cells were seeded in a 15cm dish.

[0180] Viral titer detection. Viral titer was determined by measuring p24 using an enzyme-linked immunosorbent assay (ELISA) with the QuickTiter® lentiviral titer kit (Cell Biolabs, catalog number VPK-107). Viral particles were precipitated before ELISA analysis to prevent detection of soluble p24 protein, as per the manufacturer's instructions.

[0181] Viral RNA isolation and quantification. Viral RNA was isolated using two methods. In one method, viral particles were centrifuged at 120,000 g for 90 minutes, and then the viral RNA was purified using the miRNeasy Mini kit (QIAGEN®, catalog number 217004). Alternatively, viral RNA was directly isolated from 140 μl of viral supernatant using the QIAamp Viral RNA Mini kit (QIAGEN®, catalog number 52904). Reverse transcription of viral RNA was performed using the QuantiTect® reverse transcription kit (QIAGEN). TaqMan® probes, specifically designed for quantitative PCR (QPCR), were used. Quantification was performed by QPCR or digital PCR. Absolute QPCR using the standard curve method was performed using the SYBR® Green qPCR master mixture (ThermoFisher). Standard curves were prepared using 50,500, 5000, 50000, and 500000 copies of pSacas9-HBB-sgRNA1 (for SaCas9 and sgRNA) or pCDH-GFP (for EGFP) plasmid DNA. The concentrations of plasmid DNA used to prepare the standards were determined using a NanoDrop® 2000 (catalog number ND-2000) spectrophotometer (ThermoFisher). PCR was performed using an ABI7500 instrument (Applied Biosystems, ThermoFisher). Dissociation analysis was performed after amplification to determine the specificity of the PCR product (amplification was specific if only one peak was observed in the dissociation curve). Alternatively, digital PCR was performed for quantification using a QuantStudio® 3D Digital PCR system (ThermoFisher Scientific).

[0182] Lentivirus-like particles and lentivirus particle transduction. Various amounts of concentrated virus particles (equivalent to 10-200 ng of p24 protein) were introduced into GFP-reporter cells (2 × 10⁶). 4The virus was added to cells and grown in 24-well plates with 8-12 μg / ml of polyblen in DMEM / 10% FBS. Using the supernatant containing unenriched virus, GFP-reporter cells were transduced using half fresh medium and half virus-containing supernatant. The cells were incubated with virus-containing medium for 12 hours, after which the medium was replaced with fresh DMEM medium containing 10% FBS.

[0183] Extraction of genomic DNA from human cells. Genomic DNA was isolated from cells using the QIAamp DNA Mini kit (Qiagen, Germantown, Maryland) according to the manufacturer's instructions.

[0184] Western blotting. Lentivirus or lentivirus-like particles (200 ng) were directly dissolved in 50 μl of 1× SDS loading buffer and heated at 95°C for 5 minutes. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted for MA(p17) using anti-p17 antibody (Fisher Scientific, catalog no. PA14954, 1:1000), for CA(p24) using anti-p24 antibody (Cell Biolabs, catalog no. 310810, 1:1000), and for NC using anti-p15 antibody (Abcam, catalog no. ab66951, 1:1000). Horseradish peroxidase (HRP) conjugated secondary antibodies and chemiluminescent reagents were purchased from ThermoFisher Scientific. A Fujifilm LAS-3000 system was used to acquire Western blotting images. Protein band densitometry was analyzed using ImageJ software. (Example 2) Fusion of MS2 coat protein (MCP) to lentiviral protein for SaCas9 mRNA packaging.

[0185] The objective was to package SaCas9 mRNA into lentivirus-like particles using high-affinity interactions between bacteriophage coat proteins and their respective RNA aptamers. Aptamer-binding proteins were fused with various lentiviral proteins. The corresponding aptamer sequences were added to the 3' untranslated region (UTR) of SaCas9 (see Figure 2). Four different lentiviral proteins were tested as fusion recipients of the commonly used aptamer-binding protein, MS2 coat protein (MCP). These fusion proteins were the HIV accessory protein viral protein R (VPR) (Wu, Liu et al., J. Virol. 69(6):3389-3398 (1995)) and negative regulators, as well as the HIV structural proteins nucleocapsid protein (NC) and matrix protein (MA) (referred to as MCP×2-VPR, NEF-MCP×2, NC-MCP×2, and MA-MCP×2, respectively). The NEF protein contained three mutations (G3C, V153L, and G177E) to increase its integration into lentivirus-like particles, as described in Muratori, C., et al., Methods Mol Biol 2010; 614: 111-124. Following the published studies Wu, X., et al., J Virol 1995; 69(6): 3389-3398 and Muratori, C., et al., Methods Mol Biol 2010; 614: 111-124, two tandem copies of MCP were fused to the N-terminus of VPR and the C-terminus of NEF. For the NC fusion protein, two copies of MCP were fused to the packaging plasmid pMDLg / pRRE D64V (Third generation packaging plasmid) and psPAX2 D64V(Second-generation packaging plasmid) was inserted after the NC zinc finger 2 of the Gag precursor protein, and the NC / P1 protease cleavage site was preserved. For the MA fusion protein, amino acids 44 - 132 of MA in these packaging plasmids were replaced with 2 copies of MCP. It is known that deleting this region in MA does not interfere with virus particle assembly or budding (Jalaguier, P., et al., PLoS One 2011; 6(11): e28314). Fusing MCP with MA or NC did not disrupt the reading frame of the Gag precursor protein or the protease processing site.

[0186] Expressing MCP×2-VPR or NEF-MCP×2 during lentiviral vector production, or fusing MCP with NC or MA, did not impair the efficiency of lentiviral particle production. P24 ELISA showed that in these situations, lentiviral production was as efficient as virus production by a packaging plasmid without MCP fusion (Table 5). The control used was an unmodified packaging plasmid without MCP fusion. The plasmid name is pspAX2-D64V. Table 5. Viral production efficiency and gene editing activity of packaging plasmids with various fusion proteins

Table 5

[0187] Next, the effects of these fusion proteins on gene editing were evaluated. One copy of the MS2 aptamer was used to create SaCas9 mRNA (SaCas9 mRNA), as shown in Table 2. 1xMS2 It was added after the stop codon of ). Lentivirus-like particles were prepared and SaCas9 was used as described in Example 1. 1xMS2 This was packaged with SaCas9. 1xMS2 mRNA was transcribed in HEK293T cells during lentivirus-like particle production in the presence of any one of four different fusion proteins (see Table 4 for the method of virus particle production). As described in Example 1, unenriched virus-like particles (recovered medium) were co-transduced into GFP-reporter cells using a lentiviral vector expressing human beta-hemoglobin (HBB) sgRNA1 (plasmid number 35, pFCK-HBB-sgRNA1). Gene editing in these reporter cells resulted in insertions or deletions (indels) in the HBB target sequence inserted after the EGFP start codon, which restored the EGFP reading frame. Virus-like particles produced from the unmodified packaging plasmid contained approximately 2.5% GFP. + Reporter cells were produced. Virus-like particles expressing VPR-MCP, NEF-MCP, and MA-MCP fusion proteins GFP + Although it did not significantly increase reporter cells, the NC-MCP fusion showed more than 5% GFP. +Reporter cells were generated (see Table 5). Only one-third of the indels in the reporter cells could recover the EGFP reading frame, so only one-third of gene editing events were detectable using these reporter cells (see Javidi-Parsijani, P., et al., PLoS One 2017; 12(5): e0177444). Therefore, unenriched lentivirus-like particles expressing the NC-MCP fusion protein were able to generate indels in over 15% of the reporter cells (a detected rate of 3 × 5%). Furthermore, GFP + Cells were only observed when the lentivirus-like particle was used in conjunction with a lentiviral vector expressing HBB sgRNA1 for co-transduction. Therefore, gene editing events were limited to cells transduced by both the fusion protein lentivirus-like particle and the viral particle containing HBB sgRNA1. Thus, the rate of gene editing that occurs when NC-MCP fusion protein particles are used in the system is likely to be greater than 15%. Considering these results, further experiments were conducted using NC as the recipient protein for fusion with RNA-binding aptamer proteins.

[0188] The performance of packaging plasmids containing one or two copies of MCP fused to NC was compared (plasmid numbers 11 and 13). SaCas9 containing virus-like particles produced by NC-MCP×1 packaging plasmids was also tested. 1xMS2 The NC-MCP×1 packaging plasmid produced more GFP+ cells in the GFP reporter assay than particles produced with the NC-MCP×2 packaging plasmid (NC-MCP×1: 6.2%±0.1%, N=4; NC-MCP×2: 4.8%±0.4%, N=4; p<0.05). Considering these results, the NC-MCP×1 packaging plasmid was used in subsequent experiments unless otherwise noted.

[0189] The lentiviral Env protein VSV-G promotes extracellular vesicle production (Rolls, MM, et al., Cell 1994; 79(3): 497-506), so rather than packaging into enlarged lentiviral-like particles, SaCas9 is produced from extracellular vesicles. 1xMS2 It was possible that the mRNA could be introduced into reporter cells. To test this, we conducted experiments comparing the gene editing rates induced by a control prepared using a lentivirus-like particle expressing NC-MCP×1 (using psPAX2-D64V-NC-MS2, plasmid number 11) or a plasmid expressing mRuby (pKanCMV-mRuby3-10aa-H2B; Addgene catalog number 74258) (mRuby is a red fluorescent protein) that did not express any lentivirus packaging protein. The plasmid expressing mRuby does not express any lentivirus packaging protein and therefore does not produce a lentivirus on its own. If extracellular vesicles, rather than lentivirus-like particles, are the structures that mediate Cas9 mRNA transfer, then the mRuby mRNA or plasmid should be incorporated into the extracellular vesicles as well, and the mRuby protein should be expressed with similar efficiency to the Cas9 protein. The particles were produced as described in Example 1 and concentrated by ultracentrifugation, which was expected to precipitate both the lentivirus-like particles and extracellular vesicles. To measure gene editing, the precipitate was transduced into GFP reporter cells using lentivirus particles containing HBB sgRNA1. Particles produced using the NC-MCP modified packaging plasmid showed more than 10% GFP. + While reporter cells were generated, particles produced using the mRuby plasmid instead showed less than 1% GFP. + Reporter cells and less than 1% mRuby +Only reporter cells were generated. The precipitate containing lentivirus-like particles with NC-MCP×1 also contained Cas9 mRNA, resulting in a high GFP+ reporter cell rate. Without the NC-MS2 packaging plasmid (replaced by the mRuby plasmid), the precipitate contained only extracellular vesicles, resulting in a low GFP+ reporter cell rate. This indicated that extracellular vesicles contained little Cas9 mRNA or mRuby mRNA (mRuby was also present in a low percentage). Since VSV-G was expressed under both conditions, the key difference was whether or not the modified NC-MCP packaging plasmid was used. The results showed that extracellular vesicles were GFP+ in this system. + This suggests that it does not play a crucial role in the generation of reporter cells.

[0190] To test whether GFP+ reporter cells can be easily generated from the plasmid DNA remaining after lentivirus-like particle production, SaCas9 was used during lentivirus-like particle production using the NC-MCP modified packaging plasmid (plasmid number 11). 1xMS2 Plasmid expressing both mRNA and HBB sgRNA1 (plasmid number 40, pSaCas9) 1xMS2 Packaging cells were transfected with HBB-sgRNA1. sgRNA1 does not contain an aptamer sequence. Therefore, SaCas9 1xMS2 Both mRNA and HBB sgRNA1 are transcribed from the plasmid, but SaCas9 1xMS2 Only mRNA could be packaged into lentivirus-like particles with detectable efficiency. The resulting lentivirus-like particles were transduced into GFP reporter cells using either alone (Condition 1) or in combination with lentivirus particles containing HBB sgRNA1 (constructed using plasmid number 35) (Condition 2). In Condition 2, 10% of GFP+ reporter cells were observed, while in Condition 1, less than 3% of GFP+ cells were observed. pSaCas9 was then introduced into GFP-reporter cells. 1xMS2By simply transfecting with HBB-sgRNA1 DNA, over 10% of GFP reporter cells were generated. This data suggests that the plasmid DNA remaining after lentivirus-like particle production was SaCas9 1xMS2 This suggests that mRNA is not the primary contributor, and SaCas9 1xMS2 This supports the conclusion that mRNA is packaged within lentivirus-like particles. (Example 3) Effect of MS2 aptamer on SaCas9 mRNA stability

[0191] Two possible mechanisms exist for SaCas9 0xMS2 This may contribute to the packaging of mRNA into lentiviral-like particles. Firstly, as described by Rulli, SJ, et al., J Virol 2007; 81(12): 6623-6631, cellular mRNA (including SaCas9 mRNA) may be nonspecifically packaged within lentiviral-like particles. Secondly, SaCas9 mRNA may have several RNA structures to which NC-MCP fusion proteins or other lentiviral proteins can bind. SaCas9 1xMS2 or SaCas9 0xMS2 When the genome editing activity of unenriched lentivirus-like particles containing either of these was compared, the percentage of GFP+ cells generated by the two showed little difference (9.4% ± 0.1%, N=4; 9.1% ± 0.2%, N=4; p=0.17). Although the aptamer is not essential for the packaging of SaCa9 mRNA into lentivirus-like particles, the NC-MCP fusion significantly enhanced the packaging of SaCas9 mRNA by a factor of 10 compared to the amount of SaCas9 mRNA packaged by the unmodified packaging plasmid.

[0192] To investigate the effect of MS2 aptamers on SaCas9 mRNA levels in cells, experiments were conducted to evaluate the effects of 0, 1, 2, 3, and 12 copies of MS2 aptamers (plasmid numbers 15-19) attached to the 3' end of SaCas9 mRNA on mRNA levels. n×MS2 Equal amounts of plasmid DNA encoding (n represents 0, 1, 2, 3, or 12) were transfected into HEK293T cells, and the steady-state levels of SaCas9 mRNA were compared by real-time RT-PCR. Addition of one aptamer after the SaCas9 coding sequence slightly reduced the steady-state level of SaCas9 mRNA, while addition of two or more aptamers significantly reduced the steady-state level of SaCas9 mRNA (Table 6). Consistent with the decrease in mRNA levels, SaCas9 n×MS2 When a plasmid encoding (n indicates 0, 1, 2, 3, or 12) (250 ng) is co-transfected with a plasmid expressing HBB sgRNA1 (250 ng) in GFP reporter cells (24-well plate), one aptamer will result in the observed GFP. + While the percentage of reporter cells was slightly reduced, more aptamers further reduced the observed number (Table 6). Flow cytometry and qRT-PCR were performed 72 hours after transfection. HBB sgRNA1 was used as a control for transfection efficiency in qRT-PCR. These data suggest that the addition of the MS2 aptamer reduced SaCas9 stability. Since SaCas9 mRNA, once released from lentivirus-like particles, is not replaceable (regenerative), this reduction in mRNA stability may have a measurable effect on genome editing activity. Table 6. Effects of MS2 aptamers on SaCas9 expression [Table 6] The mean ± standard error of four iterations is shown. #: p<0.0001 compared to no aptamer;##, p<0.0001 compared to one aptamer;*, p<0.05 when one aptamer is compared to two aptamers;**, p<0.01 when no aptamer is compared to one, two, three or twelve aptamers. (Example 4) The human HBB gene 3'UTR sequence increased SaCas9 mRNA stability.

[0193] SaCas9 1xMS2 Experiments were conducted to evaluate whether the addition of one or two copies of the human HBB gene 3'UTR sequence after the MS2 aptamer of plasmid pSaCas9 could enhance SaCas9 mRNA stability and translationability in the presence of the aptamer. 1×ms2 and pSaCas9 1×ms2 -2×3'UTR was transfected into HEK293T cells (plasmid numbers 16 and 36). Steady-state levels of SaCas9 mRNA in the cells were assessed by real-time RT-PCR. Addition of two human HBB gene 3'UTR sequences was found to increase the steady-state level of SaCas9 mRNA by 30% (1.0±0.03 vs. 1.3±0.08, n=4, p<0.05). SaCas9 was then added to GFP reporter cells. 1×MS2 Lentivirus-like particles containing -2×3'UTR mRNA or SaCas9 1×MS2 Simultaneous transduction was performed using lentivirus-like particles containing mRNA (without stabilizing sequence) and lentivirus particles containing HBB sgRNA1 (prepared using plasmid number 35). SaCas9 1×MS2 Transduction using lentivirus-like particles containing -2×3'UTR mRNA is performed by SaCas9 1×MS2 GFP is more effective than lentivirus-like particles containing mRNA. + This resulted in a significant increase in the percentage of reporter cells (see Figure 3). Therefore, SaCas9 with two copies of the HBB 3'UTR sequence was found to be involved. 1×MS2 The addition of this feature improved the system's performance. (Example 5) SaCas9 lentivirus-like particles based on the PP7 coated protein and its aptamer showed high genome editing activity.

[0194] PP7 coat protein (PCP) is another RNA aptamer-binding protein (see Lim, F., et al., J Biol Chem 2001; 276(25): 22507-22513 and Wu, B., et al., Biophysical Journal 2012; 102(12): 2936-2944). Experiments were conducted to compare the usefulness of the PP7 protein and its forcing aptamer sequence in this system as a substitute for MCP and its aptamer sequence. MCP was replaced with PCP, thereby producing a packaging plasmid that creates an NC-PCP fusion. Additionally, a SaCas9 mRNA expression plasmid (plasmid number 20, pSaCas9) was created in which the MS2 aptamer coding sequence was replaced with the PP7 aptamer coding sequence. 1×PP7 ) were also constructed. Using these plasmids, SaCas9 1×PP7 We created lentivirus-like particles packaged with mRNA using SaCas9. 1×PP7 When lentivirus-like particles containing SaCas9 are used to simultaneously transduce GFP reporter cells using a lentiviral vector expressing HBB-targeted sgRNA1, SaCas9 1×MS2 More GFP+ reporter cells were obtained than with lentivirus-like particles containing the specified substance (9.4%±0.14%, N=4; 8.1%±0.04%, N=4, p<0.01).

[0195] As summarized in Table 7, the amount of SaCas9 mRNA packaged in lentivirus-like particles was evaluated by real-time RT-PCR. Particle types included lentiviruses (column 2), lentivirus-like particles produced by packaging plasmids without ABP fusions (column 3), lentivirus-like particles produced by NC-MCP fusion packaging plasmids (columns 4-6), and lentivirus particles produced by NC-PCP fusion packaging plasmids (columns 7-9). SaCas9 mRNA was either aptamer-free (columns 2, 3, 4, and 7), contained one aptamer (columns 5 and 8), or contained one aptamer and two copies of HBB 3'UTR (columns 6 and 9). RNA was purified from various lentiviruses or lentivirus-like particles containing 200 ng of p24. Equal amounts of GFP-lentivirus were included in each sample to normalize RNA purification, RT, and PCR. Copy number estimation was based on the assumption that each lentivirus particle contained two copies of the lentivirus genome. By comparing SaCas9 mRNA levels in equal amounts of SaCas9-expressing lentiviruses (known to have 2 copies of genome per particle) or lentivirus-like particles, the inventors calculated the average copy number of SaCas9 mRNA per particle under each condition. Table 7 shows the mean ± standard error, with the number in parentheses being the number of repeats. Table 7. Comparison of SaCas9 copy numbers in various lentivirus-like particles using QRT-PCR. [Table 7]

[0196] It was found that without an ABP fused to the NC and without an aptamer after the SaCas9 stop codon, lentivirus-like particles had fewer than two copies of SaCas9 mRNA per particle. This may be a result of the random packaging of highly expressed cellular RNA by the virus-like particles. With an ABP fused to the NC (MCP or PCP), the SaCas9 mRNA copy number per particle increased 10-20 times, regardless of whether an aptamer sequence was added to SaCas9. Addition of the HBB 3'UTR tripled the copy number when both ABPs were present, which is most likely a result of increased RNA stability before and after packaging. Adding a single aptamer to SaCas9 mRNA did not increase the copy number of SaCas9 mRNA per particle compared to SaCas9 mRNA without the aptamer. This is most likely due to a combination of factors: the aptamer increases the binding of SaCas9 mRNA to the NC-ABP fusion protein but decreases the stability of SaCas9 mRNA. These effects of aptamers on SaCas9 mRNA are also consistent with the observation that HBB 3'UTR addition increases the copy number of SaCas9 mRNA per particle, as HBB 3'UTR does not increase binding to ABP but increases mRNA stability.

[0197] The expression of viral proteins MA, CA, and p15 (from which NC is processed) was also evaluated by Western blotting in a normal GFP lentiviral vector, as well as in MCP and PCP-based lentivirus-like particles. The NC-PCP fusion protein was found to migrate as a single band with a predicted size of approximately 22 kDa, although a further smaller band of approximately 14 kDa was observed for the NC-MCP fusion protein, indicating partial degradation. The MA and CA proteins were observed to migrate at their predicted sizes from all particles, suggesting that insertion of MCP or PCP into the NC protein does not affect the processing of other lentiviral proteins. Electron microscopy analysis of MCP- and PCP-based lentivirus-like particles revealed similar particle sizes (see Figure 4). (Example 6) Transient expression of SaCas9 mRNA from lentivirus-like particles.

[0198] To determine the duration of SaCas9 mRNA released from lentivirus-like particles, SaCas9 1xPP7 Lentivirus-like particles containing mRNA (plasmid number 20), SaCas9 1xMS2 Lentiviral particles containing mRNA (plasmid number 16) or SaCas9 1×MS2-HEK293T cells were transduced using lentiviral particles (plasmid number 36) containing 2×3'UTR mRNA, and SaCas9 mRNA levels were measured by quantitative RT-PCR at 24, 48, 72, and 96 hours post-transduction. For control, SaCas9 mRNA levels expressed from adeno-associated virus (AAV, produced using plasmid number 15) and embedded-defective lentivirus (IDLV, produced using plasmid number 31) were also evaluated. All DNA was excluded before reverse transcription; therefore, all detected SaCas9 nucleic acids were mRNA. As shown in Figure 5A, for both AAV and IDLV, SaCas9 mRNA levels increased 48 hours after transduction, consistent with the initiation of transcription from the DNA template. SaCas9 mRNA levels in cells transduced with AAV were still higher at 96 hours post-transduction than at 24 hours post-transduction. In cells transduced using IDLV, SaCas9 mRNA levels began to decline after 72 hours, but expression at 96 hours post-transduction was still about half of that at 24 hours post-transduction. In cells transduced using lentivirus-like particles packaged with modified SaCas9, SaCas9 mRNA levels declined at 24 hours post-transduction, as shown in Figure 5B. At 96 hours post-transduction, SaCas9 mRNA levels were less than 25% of those at 24 hours post-transduction. Addition of HBB 3'UTR to SaCas9 mRNA slowed its degradation. (Example 7) Efficient genome editing activity from transient expression of SaCas9 mRNA

[0199] SaCas9 1×MS2Lentivirus-like particles containing -2×3’UTR mRNA (prepared using plasmid number 36) and lentivirus particles expressing HBB sgRNA1 (prepared using plasmid number 35) were produced and concentrated by Tangential Flow Filtration as described in Example 1. Virus concentration was determined by p24 ELISA. As described in Example 1, lentivirus-like particles and lentivirus particles (each 30 ng of p24 protein) were used to co-transduce 2.5×10 4 GFP reporter cells. As described in Example 1, HBB sgRNA1 was designed against a mutated HBB sequence known to cause sickle cell disease with a one-nucleotide mismatch to the endogenous HBB sequence in reporter cells. The target sequence of HBB sgRNA1 inserted between the first and second codons of the GFP coding sequence in the transduced GFP reporter cells was amplified, and next-generation sequencing was performed to detect indels. Sequence analysis revealed that 86.5% of the alleles had indels. Thus, the described system was highly efficient in editing the sickle cell disease mutation. By closely examining the positions of the indels, most of the indels were found to be near the predicted cleavage site (3 nt from the PAM) (see Figure 6 listing SEQ ID NOs: 103 - 112 from top to bottom), which suggests that the observed indels are from gene editing guided by HBB sgRNA1. (Example 8) sgRNA packaging into lentivirus-like particles.

[0200] Experiments were conducted to determine whether sgRNAs can also be packaged into lentivirus-like particles via interactions between coat proteins and their respective aptamers. Other studies have found that aptamer sequences can be added after the stem-loop II, tetra-loop, or 3' end of SpCas9 sgRNA (Shechner, DM, et al., Nat Methods 2015; 12(7): 664-670 and Konermann, S., et al., Nature). 2015; 517(7536): 583-588). However, there was no available information on acceptable sites for aptamer addition in SaCas9 sgRNA. Modified plasmids were generated by adding one or two MS2 aptamers to the stem-loop II (ST2), tetra-loop (tetra), or 3' end of the SaCas9 sgRNA in the plasmid pSaCas9-HBB-sgRNA1 (plasmid number 22), which expresses both SaCas9 mRNA and HBB sgRNA1. The modified plasmid DNA was transfected into GFP reporter cells. Addition of one aptamer at the 3' end of the sgRNA showed little reduction in the percentage of GFP+ reporter cells (Table 8). Considering these results, sgRNAs with aptamer 3' additions were used in subsequent experiments. Table 8. Effect of aptamer addition on sgRNA performance [Table 8] The mean ± SEM (N=3) is shown. * indicates that the results were significantly lower than the control (without aptamer) by ANOVA and Tukey's multiple comparison test (p<0.05).

[0201] Another experiment involved modifying 3'MS2 aptamer HBB sgRNA1 (HBB sgRNA1 3’MS2This was performed to evaluate whether the HBB sgRNA could be packaged into lentivirus-like particles expressing the NC-MCP fusion protein using a packaging plasmid. Plasmid DNA expressing modified and unmodified HBB sgRNA1 (plasmid number 22 for sgRNA without an aptamer, and plasmid number 24 for sgRNA with a 3'MS2 aptamer) was co-transfected into HEK293T cells during lentivirus-like particle production using an MCP-based packaging plasmid (pspAX2-D64V-NC-MCP; plasmid number 11). Real-time PCR revealed 30-fold more HBB sgRNA than unmodified HBB sgRNA after normalization by the lentivirus-like particle input. 3’MS2 It was detected. Consistent with previous data, NC-MCP×1 produced 5 times more HBB sgRNA1 than NC-MCP×2. 3’MS2 It was packaged.

[0202] To test whether SaCas9 mRNA and HBB sgRNA can be packaged simultaneously in lentiviral particles, SaCas9 1xMS2 and HBB sgRNA 3’MS2 Plasmid DNA expressing both (plasmid number 28) was co-transfected into HEK293T cells during lentivirus-like particle production using an MCP-based packaging plasmid (psPAX2-D64V-NC-MS2, plasmid number 11). The resulting lentivirus-like particles (cell size 2.5 × 10⁶) were obtained. 4 70 ng per individual Transduction of GFP reporter cells using p24) as described in Example 1 produced GFP-positive cells up to 3.6%. However, simultaneous transduction of GFP reporter cells using an HBB sgRNA1-containing lentivirus (constructed using plasmid number 35) resulted in GFP +The percentage of cells was increased by more than 10%. These experiments indicate that sgRNA can be packaged in lentivirus-like particles, but the amount of packaged sgRNA resulted in low editing efficiency. Another experiment was performed using lentivirus-like particles (constructed using plasmid number 43) packaged with sgRNA having two copies of the HBB 3'UTR sequence at the 3' end. This modification was found not to improve the packaging / function of the sgRNA. Therefore, for highly efficient gene editing, sgRNA may need to be expressed from a lentivirus or AAV. (Example 9) Lentiviral-like particle delivery of SaCas9 mRNA reduced the risk of off-target gene editing events.

[0203] HBB sgRNA1 was designed to target the Sickle mutation and has a single nucleotide mismatch to the wild-type HBB sequence. Therefore, the corresponding wild-type HBB gene sequence in GFP reporter cells may be considered "off-target" to SaCas9 / HBB sgRNA1. Experiments were conducted to determine whether the provided lentiviral-like delivery system exhibits a low off-target gene editing rate. GFP reporter cells were transduced using four different combinations of virus / virus-like particles, and the indel rate of the wild-type HBB locus in GFP reporter cells was determined and compared. The viruses or lentiviral-like particles used were as follows: Condition 1: SaCas9 1xMS2Condition 1: Lentivirus-like particles containing mRNA (prepared using plasmid number 16); Condition 2: AAV containing SaCas9 mRNA (prepared using plasmid number 15, pSaCas9); Condition 3: Integrated defective lentivirus (prepared using plasmid number 31, packaged using pspAX2-D64V (Addgene catalog number 63586) containing pCK002-HBB-sgRNA and inactive integrase), IDLV expressing both SaCas9 mRNA and HBB sgRNA1; and Condition 4: Integrated competent lentivirus (prepared using plasmid number 31, pCK002-HBB-sgRNA1, and packaged using plasmid pspAX2 (Addgene catalog number 12260) containing active integrase. For conditions 1 and 2, HBB To express sgRNA1, GFP reporter cells were co-transduced using a lentivirus (pFCK-HBB-sgRNA1, plasmid number 35) in Condition 1, or AAV (pAAV-HBB(n)-sgRNA1, plasmid number 34) in Condition 2. The results are shown in Table 9. Different percentages of GFP reporter cells were observed in all four treatments for the generation of indels during Sickle mutations in the GFP cassette of GFP reporter cells. Transduced cells were sorted by GFP activation sorting. The GFP-positive cell percentage was 89–95%, indicating the presence of functional SaCas9 / HBB sgRNA1 in the majority of cells in each of the four conditions. DNA derived from the endogenous HBB locus was amplified and sequenced by next-generation sequencing. The lentivirus-like particle system described in Condition 1 had the lowest indel rate, demonstrating that delivering SaCas9 mRNA by lentivirus-like particles for gene editing is safer than other viral delivery systems. Table 9. Indel rate at the wild-type HBB locus in GFP reporter cells [Table 9] Table 10 - Sequence List [Table 10-1] Table 10-2 Table 10-3 Table 10-4 Table 10-5 Table 10-6 Table 10-7 Table 10-8 Table 10-9 Table 10-10 Table 10-11 Table 10-12 Table 10-13 Table 10-14 Table 10-15 Table 10-16 Table 10-17 Table 10-18 (Example 10) System for packaging SaCas9 mRNA into LVLP

[0204] A system for efficiently packaging SaCas9 mRNA into LVLPs is described herein. LVLPs enable transient SaCas9 expression and highly efficient genome editing. They result in lower off-target rates compared to AAV and lentiviral delivery. The SaCas9 LVLPs described herein possess transient expression characteristics of RNP-, mRNA-, and nanoparticle-delivery strategies while retaining the transduction efficiency of lentiviral vectors. This system can be used to package mRNA encoding various editing proteins for genome editing.

[0205] Plasmids: pRSV-Rev (Addgene #12253), pMD2.G (Addgene #12259), pMDLg / pRRE (Addgene #12251), psPAX2-D64V (Addgene #63586), pSL-MS2x12 (Addgene #27119), pKanCMV-mRuby3-10aa-H2B (Addgene #74258), and pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (Addgene #61591) were purchased from Addgene. pCDH-GFP was purchased from SBI (CD513B-1). Other plasmids were generated (see Table 2). Gene synthesis was performed by GenScript Inc. All generated constructs were sequenced. Sequence information for primers, oligos, and synthetic DNA fragments is shown in Table 10.

[0206] GFP Reporter Assay for Gene Editing Activity: The EGFP reporter cell line described by Javidi-Parisjani et al. was used to detect the gene editing activity of SaCas9 / human beta-hemoglobin (HBB) sgRNA1 at the target sequence inserted into the GFP reporter cassette. GFP reporter cells (derived from HEK293T cells) did not express EGFP due to disruption of the EGFP reading frame by an HBB sickle mutation between the start and second codons of the EGFP coding sequence and insertion of the IL2RG target sequence. Indels formed after gene editing could restore the EGFP reading frame, resulting in EGFP expression. GFP-positive cells were analyzed by fluorescence microscopy or flow cytometry (BD Biosciences, Accuri C6). Single-cell suspensions were prepared in PBS / 0.5% FBS for analysis. Cells without fluorescent protein expression were used as negative controls, and the marker was positioned so that 99.9% of cells were to the left of the marker. In treated samples, cells to the right of the marker were considered positive.

[0207] AAV6 virus production and transduction: Adeno-associated viruses expressing SaCas9 and HBB sgRNA1 were generated from AAV vectors pSaCas9 (expressing SaCas9) and pSaCas9-HBB-sgRNA1 (expressing HBB sgRNA1 and containing a donor template for homologous recombination that modifies the wild-type HBB gene with a Sickle mutation), respectively. AAV serotype 6 (AAV6) production and enrichment were performed by Virovek, Inc. (Hayward, CA). For AAV6 transduction, cells were changed to serum-free medium or OPTI-MEM, and AAV6 was transduced to the viral genome 10 3 ~10 4 The solution was added to cells at individual / cell titers. After 24 hours, the culture medium was changed to serum-containing growth medium.

[0208] Lentivirus and LVLP Production Lentiviruses were produced using second or third-generation packaging systems as previously described by Javidi-Parisjani et al. The lentivirus vector pCK002-HBB-sgRNA1, expressing both SaCas9 and HBB sgRNA1, was used to produce incorporated competent lentiviruses (packaged by packaging plasmids pspAX2 or pMDLg / pRRE) and incorporated defective lentiviruses (IDLVs) packaged by packaging plasmids pspAX2-D64V or pMDLg / pRRE-D64V. To produce LVLPs packaged with SaCas9 mRNA, HEK293T cells were transfected with a mixture of DNA from the packaging plasmid (NC-MCP or NC-PCP modification), envelope plasmid (pMD2.G), and SaCas9 expression plasmid in the DNA ratios shown in Table 11. When VPR or NEF fusion proteins were used for packaging, SaCas9 mRNA and plasmid DNA were also included for their expression. Transfection was mediated by polyethyleneimine (PEI, Polysciences Inc.) in a DNA:PEI ratio of 1:2. Cell culture and DNA transfection were carried out as described by Javidi-Parisjani et al. 24 hours after transfection, the medium was changed to Opti-MEM, and lentiviruses or LVLPs were collected twice, once every 24 hours. The supernatant was rotated at 500 g for 10 minutes to remove cell debris before further processing as described below. Table 11. Plasmid DNA used to transfect HEK293T cells to produce lentiviral particles loaded with SaCas9 mRNA. a [Table 11-1] [Table 11-2] a13 x 10 cells 24 hours before transfection 6 I sowed the seeds in a 15cm dish.

[0209] Lentivirus and LVLP Concentration Three methods were used to concentrate lentiviruses and LVLPs: 1) The supernatant containing the virus or LVLP was placed on a 10 ml 20% sucrose cushion and then centrifuged at 20,000 g, 4°C, for 4 hours; 2) The supernatant containing the virus or LVLP was mixed with Lenti-X® Concentrator (Takara, catalog no. 631232) in a 3:1 (volume / volume) ratio, incubated at 4°C, for 30 minutes, and then centrifuged at 4°C, 1,500 × g, for 45 minutes; 3) The supernatant was concentrated using the KR2i TFF System (KrosFlo® Research 2i Tangential Flow Filtration System) (Spectrum Lab, catalog no. SYR2-U20) in concentrate-diafiltration-concentrate mode. Typically, 150–300 ml of supernatant was first concentrated to approximately 50 ml, diafiltration with 500 ml to 1000 ml of PBS, and finally concentrated to approximately 8 ml. Hollow fiber filter modules were prepared from modified polyethersulfone with a molecular weight cutoff of 500 kDa. Flow rate and pressure limits were 80 ml / min and 8 psi for filter module D02-E500-05-N, and 10 ml / min and 5 psi for filter module C02-E500-05-N. Since the TFF method produced lentiviruses and LVLPs with the best activity, data were obtained using viruses or LVLPs concentrated by the TFF system unless otherwise noted.

[0210] Lentivirus and LVLP quantitative analysis: Viral titer measured using p24-based ELISA (Cell Biolabs, QuickTiter®). The determination was made according to Kit catalog number VPK-107. When assaying unpurified samples, the virus particles were precipitated according to the manufacturer's instructions for use so that the soluble p24 peptide would not be detected.

[0211] Transmission electron microscopy was performed at the Cellular Imaging Shared Resource of Wake Forest Baptist Health Center (Winston-Salem, NC). For negative staining, 30 ml of virus-containing supernatant was concentrated to approximately 1 ml using ultracentrifugation. Then, a plain carbon grid was immersed in 20 μl of the virus sample, and the particles were stained with phosphotungstic acid. The sample was dried and observed under an FEI Tecnai G2 30 electron microscope (FEI, Hillsboro, OR).

[0212] Western blot analysis: To analyze viral proteins from lentiviruses and LVLPs, purified lentiviruses or LVLPs (200 ng p24, by ELISA) were dissolved in 20 μl of 1× Laemmli sample buffer. Proteins in each sample were separated on an SDS-PAGE gel and analyzed by Western blotting. Antibodies used included HIV1 p17 antibody for MA (ThermoFisher Scientific, catalog number PA1-4954, 1:1000), HIV1 p15 polyclonal antibody for NC (Abcam, catalog number ab66951, 1:1000), and p24 monoclonal antibody for CA (Cell Biolabs, catalog number 310810, 1:1000).

[0213] To detect the SaCas9 protein, HEK293T cells or GFP reporter cells were transfected with SaCas9-expressing plasmid DNA, or co-transduced using an IDLV expressing 100 ng SaCas9 mRNA package LVLP and 100 ng p24 HBB sgRNA1. 48 hours after transfection or transduction, cells were lysed with 100 μl of 1×Laemmli sample buffer, and equal volumes of each sample were loaded for SDS-PAGE separation and Western blot analysis with anti-HA antibody (ProteinTech, 51064-2-AP, 1:1000) and anti-Cas9 antibody (Millipore Sigma, MAB131872, clone 6F7, 1:1000) to detect HA-tagged SaCas9. Anti-beta-actin (Sigma, A5441, 1:5000) was used to compare input levels.

[0214] HRP-conjugated anti-mouse IgG(H+L) (ThermoFisher Scientific, catalog number 31430, 1:5000) and anti-rabbit IgG(H+L) (catalog number 31460, 1:5000) secondary antibodies were used in Western blotting. A chemiluminescent reagent (Pierce) was used under the LAS-3000 system (Fujifilm) to visualize the protein signal.

[0215] RNA Isolation from Lentiviruses or LVLPs: The miRNeasy Mini Kit (QIAGEN catalog number / ID: 217004) was used to isolate RNA from enriched lentiviruses or LVLPs. Alternatively, RNA was directly isolated from 140 μl of particle-containing supernatant using the QIAamp Viral RNA Mini Kit (QIAGEN).

[0216] For RNA RT-PCR analysis, the QuantiTect Reverse Transcription Kit (QIAGEN) was used to reverse transcribe RNA into cDNA. Custom-designed hydrolysis probes specific to SaCas9, HBB sgRNA1, and EGFP (ThermoFisher Scientific) were used in qPCR along with the TaqMan Universal PCR Master Mix (ThermoFisher Scientific). PCR was performed using an ABI 7500 instrument.

[0217] Lentivirus and LVLP Transduction: Cells grown in 24-well plates containing 8 μg / ml polyblen with varying amounts of lentivirus or LVLP (ng p24 protein) were 2.5 × 10⁶ cells. 4 The cells were added individually. The cells were incubated in particle-containing medium for 12-24 hours, and then replaced with normal medium.

[0218] For gene editing in human cells using Cas9 expressed from AAV6, GFP reporter cells were transduced using SaCas9-expressing AAV6 and HBB sgRNA1-expressing AAV6. For gene editing using LV or IDLV (packaged with a packaging plasmid containing a D64V mutation in the integrase) expressing both SaCas9 and HBB sgRNA1, 2.5 × 10⁶ cells were transduced in a 24-well plate using 10–300 ng of virus equivalent to p24. 4 It was used for individual transduction. For gene editing using LVLP, various amounts of SaCas9 mRNA-containing LVLP were simultaneously transduced into HEK293T cells or GFP reporter cells using IDLV expressing HBB sgRNA1. 48–72 hours after transduction, gene editing activity was analyzed by GFP reporter assay or next-generation sequencing.

[0219] To investigate gene editing in human lymphoblastoid cells immortalized by Epstein-Barr virus transformation, we purchased the human lymphoblastoid cell line GM16265 from the Corrie Institute. Lymphoblasts were cultured in RPMI 1640 containing 2 mmol / L L-glutamine and 15% fetal bovine serum at 37°C under 5% carbon dioxide. For LVLP and IDLV transduction, 2 × 10⁶ cells were used. 5 Each cell was added to 0.5 ml of RPMI growth medium. Next, 100 p24 or 500 p24 SaCas9 LVLP and IL2RG sgRNA IDLV were added to the cells. Polybrene was added to the medium at a final concentration of 8 μg / ml. Fresh medium was replaced 20 hours after transduction. Cells were harvested 72 hours after transduction for DNA analysis by next-generation sequencing.

[0220] Next-Generation Sequencing and Data Analysis The following DNA regions were amplified for next-generation sequencing analysis: 1) endogenous HBB target sequence, 2) endogenous IL2RG target sequence, and 3) HBB target sequence in an integrated GFP expression cassette. To amplify the endogenous HBB target sequence for sequencing, a nested PCR strategy was used to avoid amplifying sequences from the viral vector template. First, primers HBB-1849F and HBB-5277R were used to amplify a 3.4kb region derived from the HBB gene locus. These two primers cannot amplify sequences derived from the template in the viral vector. Next, two internal primers, HBB-MUT-F and HBB-MUT-R, were used to amplify the target DNA for sequencing (Table 10; SEQ ID NOs: 34-40). To amplify the endogenous IL2RG target sequence, primers IL2RG-1029F and IL2RG-3301R were used to amplify the target region derived from treated cells (sequence amplification from the template in the viral vector was not possible). Then, for sequencing, two internal primers IL2RG-mut-F1 and IL2RG-mut-R4 were used to amplify the target DNA derived from the first PCR product. For sequencing, reporter-mut-F and reporter-mut-R primers were used to amplify the HBB target sequence derived from the integrated EGFP reporter (Table 10; SEQ ID NOs. 98-102). KAPA Proofreading HotStart® ReadyMix from Biosystems (Wilmington, MA) was used for PCR. To increase sequence diversity during next-generation sequencing, different numbers of staggered nucleotides were added after the 5' primer barcode to amplify the same target from different samples. Single-end reads from the 5' of the PCR product were obtained by next-generation sequencing using an Illumina NextSeq 500 as described by Javidi-Parisjani et al. Low-quality reads were removed. All sequences were screened based on barcodes.

[0221] After removing the 3' linker and 5' barcode sequences, the obtained read data was sent to online Cas-Analyzer software for mutation analysis. When a reference sequence was sent, primer sequences were excluded if they were at least 12nt away from the sgRNA target. Otherwise, primer sequences were included in the reference. Only read data containing both the 5'12nt and 3'12nt of the reference sequence were included in the indel analysis. The total indel rate was the difference between 1 and the percentage of read data that did not contain mutations. The top 8–10 most frequently observed read data are shown.

[0222] SaCas9 mRNA degradation analysis: To compare SaCas9 RNA levels expressed from IDLV and LVLP, HEK293 cells 2.5 × 10⁶ 4 Each cell was transduced using IDLV or LVLP containing 35 ng p24. IDLV particles could express SaCas9, and LVLPs were packaged containing SaCas9 mRNA. 24 hours after transduction, cells were maintained in a medium containing 0.5% FBS to restrict cell division. Fresh medium was changed every 48 hours. Cells were harvested 24, 48, 72, and 96 hours after transduction, followed by RNA extraction and RT-qPCR analysis. Three hydrolysis probes, SaCas9, GAPDH, and RPLP0, were used for expression analysis. Relative Cas9 mRNA levels of different particles 24 hours after transduction were determined by normalization to RPLP0. Since no new mRNA was generated after transduction, including in LVLPs, normalization was not performed when evaluating mRNA degradation of Cas9 mRNA from the same particle to avoid potential cell proliferation interference in RNA degradation analysis. However, RPLP0 and GAPDH expression were examined for all samples to confirm that sample preparation was successful and consistent.

[0223] Statistical analysis was performed using GraphPad Prism software (version 5.0, GraphPad Software Inc). A t-test was used to compare the means of two groups. Tukey's post-hoc test was performed after analysis of variance (ANOVA) for data from more than two groups. In the case of two factors, Bonferroni post-hoc test was performed after ANOVA. A p < 0.05 was considered statistically significant.

[0224] Fusing a nucleocapsid (NC) protein with an RNA-binding protein enables efficient packaging of SaCas9 mRNA into lentiviral-like particles. To package SaCas9 mRNA into LVLPs via specific MS2 / MCP interactions, the MS2-binding protein MCP was fused to the lentiviral protein, and MCP interacting with the MS2 aptamer was added after the stop codon of the SaCas9 mRNA. To find the MCP fusion partner with the most efficient mRNA packaging, four different lentiviral proteins—viral protein R (VPR), negative regulator (NEF), nucleocapsid protein (NC), and matrix protein (MA)—were explored as recipient proteins for MCP based on lapses: 1) mutant NEFs (including three mutations: G3C, V153L, and G177E) can be incorporated into lentiviral particles up to 1100 molecules / capsid and used as a vehicle for foreign protein delivery; 2) VPRs can be incorporated into the viral core up to 550 copies / particle and similarly used as a protein delivery vehicle; 3) NCs, processed from one of each of the 2500-5000 Gag precursors forming the lentiviral particle, are the main RNA-binding protein in the viral core; and 4) MAs, also processed from Gag proteins, have been shown to bind to tRNA.

[0225] MCP was fused with NEF and VPR in the configurations shown in the duplicated diagrams Figure 2 and Figure 7A. MCP-VPR and NEF-MCP were expressed from plasmid DNA co-transfected with the unmodified packaging plasmid during LVLP production (Figure 7B). To fuse MCP with NC, the packaging plasmid was modified by inserting the MCP coding sequence after the coding sequence of the second zinc finger of NC. Since the second zinc finger of NC is necessary for virion production, and deletion of this domain reduced virion production by a factor of ten, this domain was conserved rather than replaced with MCP. To fuse MCP with MA, the coding sequence for MA amino acids 44-132 was replaced with that of MCP in the packaging plasmid, as this region of MA is not necessary for virus production. The MCP coding sequence was inserted into either NC or MA of the packaging plasmid so that the Gag precursor protein reading frame and protease processing site were conserved.

[0226] To investigate whether MCP incorporation affects virion production, we compared the efficiency of GFP lentivirus production using these constructs. Expression of MCP-VPR or NEF-MCP in packaging cells did not affect GFP lentivirus production using the original packaging plasmid. The NC-MCP modified packaging plasmid produced GFP lentivirus with similar efficiency to the original packaging plasmid, while the MA-MCP modified packaging plasmid showed reduced virus production efficiency (Figure 7C).

[0227] To obtain a simple assay for comparing genome editing activity, GFP reporter cells (derived from HEK293T cells transduced using a GFP reporter lentiviral vector) were used. These GFP reporter cells were incorporated with a GFP expression cassette, in which a Sickle mutation sequence of human beta-hemoglobin (HBB), targeted by HBB sgRNA1, was inserted between the EGFP start codon and the second codon to disrupt the reading frame. Insertions or deletions (indels) in the target sequence resulting from gene editing can restore the EGFP reading frame and GFP expression. To test whether fusing MCPs to viral proteins can enhance the packaging of SaCas9 mRNA into LVLPs, SaCas9 1xMS2 SaCas9 mRNA (containing one copy of the MS2 aptamer after the stop codon) was expressed during LVLP generation using the original packaging plasmid (with or without MCP-VPR or NEF-MCP expression) or the MCP-modified packaging plasmid (Figure 7B). These LVLPs were then co-transduced into GFP reporter cells using an embedded defect lentivirus (IDLV) vector expressing HBB sgRNA1, which targets the Sickle mutation in human beta-hemoglobin (HBB) (32). LVLPs produced from the NC-MCP-modified packaging plasmid generated significantly more GFP reporter cells than LVLPs produced by all other strategies for MCP integration (p<0.01) (Figure 7D). Therefore, NC was used as the MCP recipient protein for further experiments.

[0228] The MCP dimer possessed RNA aptamer binding activity but lacked monomers or oligomers. When comparing the performance of packaging plasmids fused with NC to one or two copies of MCP, LVLPs generated by the packaging plasmid fused to one copy of MCP produced more GFP+ reporter cells than those fused to two copies of MCP (5.7% ± 0.1%, N=4 vs. 4.4% ± 0.3%, N=4; p<0.01). Therefore, the modified packaging plasmid fused to one copy of MCP was used in subsequent experiments.

[0229] To examine whether the observed gene editing activity was caused by nucleic acids in extracellular vesicles promoted by VSV-G, we included controls in which the NC-MCP modified packaging plasmid was replaced with a plasmid expressing mRuby fluorescent protein but not lentiviral packaging protein. Equivolutes of supernatant were co-transduced into GFP reporter cells using HBB sgRNA1-expressing IDLV, and the supernatant produced using the packaging plasmid contained up to 15% GFP. + Reporter cells were generated. On the other hand, the supernatant produced without the use of a packaging plasmid generated less than 0.5% GFP+ reporter cells under all conditions (Figure 7E), and the mRuby positivity rate was less than 0.5% in cells treated with the highest volume of supernatant (Figure 8). Since VSV-G was present under both conditions, the data suggest that the extracellular vehicle is GFP. + The data suggested that it did not play a major role in the generation of reporter cells. The data also suggested that residual plasmid DNA contributed little to the total gene editing activity observed.

[0230] To further investigate the contribution of residual plasmid DNA from LVLP production to the generation of GFP+ reporter cells, SaCas9 1xMS2 Only the mRNA is expressed (pSaCas9 1xMS2 ) and SaCas9 1xMS2 Expresses both mRNA and HBB sgRNA1 (pSaCas91xMS2 -HBB sgRNA1) constructs were generated. As predicted, pSaCas9 1xMS2 -Transfecting HBB sgRNA1 plasmid DNA into GFP reporter cells generated significantly more GFP+ reporter cells than transfecting pSaCas9 1xMS2 plasmid DNA (Figure 7F). Since HBB sgRNA1 without the MS2 aptamer cannot be packaged, it was predicted that the LVLP prepared from the pSaCas9 1xMS2 -HBB sgRNA1 plasmid DNA would contain insufficient copies of HBB sgRNA1. Indeed, these LVLP produced significantly fewer GFP+ reporter cells when used alone for transduction than when co-transduced with IDLV expressing HBB sgRNA1 (p < 0.0001, Figure 7F). Notably in this experiment, sgRNAs non-specifically packaged into the LVLP also contributed to background activity, explaining why higher background activity was observed in Figure 7F than in Figure 7E. These observations indicate that the main contribution is not from the plasmid DNA remaining during LVLP production, supporting more efficient packaging of SaCas9 1xMS2 mRNA when using NC-MCP LVLP.

[0231] The human beta-hemoglobin (HBB) 3' untranslated region greatly improved the genomic editing activity of SaCas9 mRNA packaged into LVLP. To determine the optimal copy number of the MS2 aptamer for packaging of SaCas9 mRNA, the effects of 0, 1, 2, 3, and 12 copies of the MS2 aptamer on SaCas9 nxMS2(where n represents 0, 1, 2, 3, or 12) Equal amounts of plasmid DNA expressing this were transfected into HEK293T cells, and the steady-state levels of SaCas9 mRNA were compared by RT-qPCR. The addition of one aptamer after the stop codon of SaCas9 slightly decreased the steady-state level of SaCas9 mRNA, while the addition of more than one aptamer significantly decreased SaCas9 mRNA (p < 0.001, Figure 9A). Consistent with the decrease in mRNA, SaCas9 nxMS2 When the expression DNA was co-transfected into GFP reporter cells with the plasmid expressing HBB sgRNA1, one aptamer slightly decreased the percentage of GFP + reporter cells, while more aptamers caused further decreases (Figure 9B). Consistently, SaCas9 1xMS2 , SaCas9 2xMS2 , SaCas9 3xMS2 or SaCas9 12xMS2 When the LVLP containing was co-transduced into GFP reporter cells with the HBB sgRNA1-expressing lentivirus, at all concentrations tested, the SaCas9 1xMS2 LVLP produced the most GFP-positive cells, and the SaCas9 12xMS2 LVLP produced the least (Figure 9C).

[0232] To enhance SaCas9 mRNA stability and translatability with or without aptamers, two copies of the 3' untranslated region (UTR) sequence from the human HBB gene were added after the SaCas9 stop codon and before the MS2 aptamer. Plasmid DNA transfection in HEK293T cells showed that the steady-state level of SaCas9 1xMS2 -HBB 3'UTR was 1.3 times that of SaCas9 0xMS2 (1.3 ± 0.08 vs. 1.0 ± 0.03, n = 4, p < 0.05).

[0233] Since more than one MS2 aptamer significantly reduced the gene editing activity of LVLP, we further studied single-copy MS2 aptamers. We also studied SaCas9 mRNA (SaCas9 mRNA) containing or not containing MS2 aptamers and HBB 3'UTR. 0xMS2 SaCas9 1xMS2 SaCas9 0xMS2 -HBB 3'UTR, SaCas9 1xMS2 LVLPs containing (HBB 3'UTR) were constructed, and each type of particle was transduced into GFP reporter cells using HBB sgRNA1-expressing IDLV. Flow cytometry analysis revealed that without MCP, GFP-positive cell production from LVLPs was inefficient (dashed line in Figure 9D). In LVLPs generated with MCP, the presence of both MS2 and HBB 3'UTR resulted in the highest genome editing activity. Surprisingly, SaCas9 mRNA without the MS2 aptamer could also produce recognizable GFP-positive cells. 1xMS2 LVLP is SaCas9 0xMS2 It showed lower activity than LVLP, which is consistent with previous observations that MS2 reduces SaCas9 mRNA stability. HBB 3'UTR is SaCas9 1xMS2 While LVLP performance has been significantly improved, SaCas9 0xMS2 The LVLP did not show improvement, and another observation was consistent with previous data. These data suggest that 1) MCP is necessary for efficient packaging of SaCas9 mRNA; and 2) MS2 aptamers decrease SaCas9 mRNA stability, but they can increase SaCas9 mRNA / NC-MCP association; and 3) HBB 3'UTR increases SaCa9 mRNA stability, and the presence of MS2 and HBB 3'UTR improved SaCas9 mRNA / NC-MCP association and SaCa9 mRNA stability.

[0234] When considering the possibility of packaging dCas9 mRNA for CRISPR-mediated gene regulation, where sgRNA may need to be modified using aptamers to enhance gene regulation, more than one aptamer may be used in the same experiment. A PP7 / PP7 coated protein (PCP)-based packaging system was also developed to enable aptamer combinations. PP7 / PCP is another RNA aptamer / ABP pair used in RNA studies. The MCP is replaced with PCP in the packaging plasmid, and the MS2 aptamer is used in the SaCas9 mRNA expression plasmid (SaCas9 1xPP7 In the expression of SaCas9 mRNA, the PP7 aptamer was used to replace it. PP7 / PCP, which packages SaCas9 LVLP, was also used in conjunction with HBB sgRNA1-expressing IDLV to efficiently produce GFP+ reporter cells when co-transduced into GFP reporter cells (Figure 9D). Surprisingly, the PCP-modified packaging system could also package PP7-free SaCas9 mRNA, in which case the packaging was PCP-dependent.

[0235] GFP reporter assay data were supported by RT-qPCR analysis of SaCas9 mRNA copy number per LVLP (Figure 9E). 1xMS2 Consistent with the observation that LVLPs containing HBB 3'UTR produced the highest percentage of GFP-positive cells, they also had the highest SaCas9 mRNA copy number (approximately 50 times more RNA molecules than those contained in a lentiviral vector containing a similar p24 protein). 0xMS2 and SaCas9 1xMS2 LVLPs have similar copies of Cas9 mRNA per particle, but Cas9 1xMS2 The gene editing activity of LVLP is Cas9 0xMS2 The stability levels were consistently lower than those of LVLP (Figure 9D). This may be a result of the MS2 aptamer reducing SaCas9 mRNA stability.

[0236] Comparing LVLPs packaged by MCP / MS2 and PCP / PP7-based systems, PCP / PP7-packaged SaCas9 LVLPs exhibited better gene editing activity than MCP / MS2-packaged LVLPs when the HBB 3'UTR was not added to SaCas9 mRNA. The opposite was true when the HBB 3'UTR was added to SaCas9 mRNA (Figure 9D). 1xMS2 -HBB 3'UTR LVLP consistently demonstrated the best gene editing activity across multiple iterations.

[0237] The expression duration of mRNA delivered by LVLP was examined and compared with that of IDLV. 24 hours after transduction, SaCas9 1xMS2 -HBB 3'UTR LVLPs exhibit the highest Cas9 mRNA levels, consistent with their high mRNA copy number / particle and mRNA stability (Figure 10A). SaCas9 1xMS2 LVLPs have the lowest mRNA expression, which is consistent with their low gene editing activity. Novel Cas9 In contrast to the increase in Cas9 mRNA expression by IDLV 48 hours post-transduction, suggesting mRNA transcription, all mRNAs delivered by LVLP gradually decreased at different rates. Compared to the respective mRNA levels 24 hours post-transduction, SaCas9 1xPP7 SaCas9 1xMS2 -HBB 3'UTR showed a similar degradation rate (Figure 10B), while SaCas9 1xMS2 This showed a faster degradation rate. This is SaCas9 1xMS2 - HBB 3'UTR and SaCas9 1xPP7 In the GFP reporter assay, SaCas9 1xMS2 This also explains why the results were better than before. Therefore, SaCas9 1xMS2 -HBB 3'UTR LVLP achieved higher transient expression compared to IDLV viral vectors.

[0238] Protein expression from LVLPs was investigated. Cas9 protein expression was readily detected in overexpressing HEK293T cells or GFP reporter cells using either SaCas9 or HA (the C-terminal tag of Cas9) antibodies, but Cas9 expression was barely detectable in GFP reporter cells 48 hours after transduction with 300 ng LVLP (Figure 10C). These cells were co-transduced using HBB sgRNA1 IDLV, and 15% of them were GFP-positive by flow cytometry analysis. Although Cas9 protein was difficult to detect in these cells by Western blotting, this suggests successful gene editing in approximately 45% of the cells. The data indicate that LVLPs were able to express sufficient Cas9 protein for efficient gene editing, even though it was barely detectable by the methods used by the inventors. The off-target rate increased with increasing Cas9 protein, suggesting that relatively low levels of Cas9 protein from LVLPs also contribute to low off-target effects.

[0239] Gag precursor proteins were processed into mature proteins by sequential protease cleavage with different cleavage rates at different sites (Figure 10D). Antibodies specific to MA(p17), CA(p24), and p15 (from which NC is processed) were used with a standard GFP lentiviral vector, MCP-based SaCas9. 1xMS2 LVLP and PCP-based SaCas9 1xPP7The sizes of processed proteins in LVLPs were analyzed. MA and CA proteins from all particles showed the same predicted sizes and indicated proper cleavage at the time of analysis (Figure 10E). The anti-MA antibody detected a weak 75kDa band in MCP- and PCP-based LVLPs. However, no similar band was detected by the anti-CA or anti-p15 antibody, indicating the absence of unprocessed Gag precursor proteins. The anti-p15 antibody detected a band slightly smaller than 15kDa in GFP lentivirus. This size indicates that cleavage at sites 4 and 5 did not occur at a detectable level at the time of analysis and is consistent with their low rates. The p15 antibody detected bands between 20–25kDa in PCP- and MCP-based LVLPs, which is consistent with the predicted sizes of NC-PCP and NC-MCP fusion proteins in LVLPs (Figure 10D). In MCP-based LVLPs, an additionally small band was detected by the p15 antibody, indicating partial degradation of the p15-MCP fusion protein. These data suggest that insertion of MCP or PCP in NC had a slight effect on Gag precursor cleavage at sites 1 (MA / CA), 2 (CA / SP1), and 3 (SP1 / NC). The effect on cleavage at sites 4 (NC / SP2) and 5 (SP2 / P6) was unclear, as these sites were not cleaved even in normal LV at the time of analysis, judging from the size of the peptides detected by the p15 antibody. Electron microscopy analysis of MCP- and PCP-based lentivirus-like particles revealed particle sizes similar to those of GFP lentivirus (Figure 11).

[0240] LVLPs packaged with SaCas9 mRNA enable highly efficient genome editing. To further determine the gene editing activity of LVLPs packaged with SaCas9 mRNA, the inventors of the SaCas9 mRNA 1xMS2 - Using LVLP (30 ng p24 protein) packaged with HBB 3'UTR mRNA together with 60 ng p24 HBB sgRNA1-expressing IDLV, GFP reporter cells 2.5 × 10 4Simultaneous transduction was performed on individual cells. 48 hours after transduction, 13% of reporter cells became GFP-positive. Target DNA for HBB sgRNA1 in GFP expression cassettes was amplified and sequenced by next-generation sequencing. Overall, 86.5% of alleles had indels mainly around the predicted cleavage site 3 nt away from the PAM (Figure 12B) (Figure 12A). The data demonstrated that delivery of SaCas9 mRNA by LVLP is highly efficient in generating indels.

[0241] To examine the activity of SaCas9 LVLP at its endogenous target, we prepared IDVLs expressing sgRNA that targets human IL2RG, a mutation of which causes X-linked severe combined immunodeficiency (SCID-X1). In HEK293T cells, 30 ng p24 of SaCas9 LVLP and 60 ng of IL2RG sgRNA IDLV generated 13% indels at the target sequence (Figure 12C). Human lymphoblasts (cells 2 × 10⁶) 5 In cells, simultaneous transduction of 100 or 500 ng p24 of both particle types generated 11% or 87.6% indels in the target sequence (Figure 12D). Indel rates differed under different conditions, but the most frequently observed mutations were the same. The data demonstrated that SaCas9 LVLP could be used to target different target genes in multiple cells.

[0242] LVLPs packaged with SaCas9 mRNA showed a lower off-target rate than those with viral vectors. HBB sgRNA1 was designed to target an HBB Sickle cell mutation, which has a single nucleotide mismatch to the wild-type HBB sequence (Figure 12E). Thus, the corresponding wild-type HBB gene sequence in GFP reporter cells can be considered an "off-target" for SaCas9 / HBB sgRNA1. Indel rates at the wild-type HBB locus were compared when SaCas9 was delivered by LVLP, AAV6, IDLV, or SaCas9-expressing LV. To modulate the percentage of cells with functional SaCas9 / HBB sgRNA1, GFP-positive cells were sorted 48 hours after transduction by GFP activity sorting. At sorting (48 hours after transduction), the percentage of GFP positivity was compared for LV, LVLP, IDLV, and AAV6 (10 4 The percentages for cells treated with (vg / cell) were 11.1%, 2.1%, 8.5%, and 6.5%, respectively. Cells were treated with the same number of LV, LVLP, and IDLV particles based on p24 levels (1.25 × 10⁶ cells). 6 At approximately 750 ng p24 per cell, embedded competent LV-treated cells already showed a higher GFP positivity rate than IDLV due to sustained CRISPR / Cas9 expression. After sorting, the GFP positivity rates were 95.4%, 88.9%, 93.3%, and 90.8%, respectively, suggesting the presence of functional SaCas9 / HBB sgRNA1 in most cells under each condition. Next, DNA from the endogenous HBB locus was amplified from the sorted cells one week after sorting and subjected to next-generation sequencing. The LVLP system had the lowest indel rate at the endogenous HBB site, while LV had the highest (Figure 12E). These data demonstrate that SaCas9 mRNA delivery by LVLP for gene editing is safer than other viral delivery systems.

[0243] In the cells described above, we searched for indels in nine potential off-targets predicted by Cas-Offinder and CRISPOR. Even in LV-treated cells, no indels were detected in eight of the nine potential HBB sgRNA1 off-targets. However, for off-target 8 (chromosome 20, 33982230bp~33982337bp), slightly more indels were detected in LV-transduced cells than in LVLP-transduced cells (Figure 12F).

[0244] Since we have GFP reporter cells transduced using different particles showing different percentages of GFP-positive cells (cells used in Figure 12A and unselected cells in Figure 12E), we analyzed the relationship between the GFP-positive percentage and the indel rate of the target sequence (in the GFP expression cassette) in these cells. We found that the indel rate increased linearly with the GFP-positive percentage (Figure 13, linear regression showed r 2 (=0.9497). The data validated the GFP reporter assay described herein for comparing genome editing activity of different particle types. (Example 11) Cas9 / sgRNA RNP packaging into lentivirus-like particles

[0245] The plasmids pMD2.G (Addgene #12259), psPAX2-D64V (Addgene #63586), pCSII-EF-miRFP709-hCdt(1 / 100) (Addgene #80007), and pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (Addgene #61591) were purchased from Addgene and have already been described. The plasmids are described in Example 10 or Table 2. All generated constructs were sequenced. Sequence information for primers, oligos, and synthetic DNA fragments is provided in Table 10.

[0246] GFP Reporter Assay for Gene Editing Activity The EGFP reporter cells described above were used to detect the gene editing activity of SaCas9 / human beta-hemoglobin (HBB) sgRNA1 or SaCas9 / IL2RG-sgRNA1 on target sequences inserted into GFP reporter cassettes. GFP reporter cells did not express EGFP due to disruption of the EGFP reading frame by HBB sickle mutation between the start codon and second codon of the EGFP coding sequence and insertion of the IL2RG target sequence. Indels formed after gene editing can restore the EGFP reading frame and result in EGFP expression. GFP-positive cells were examined using fluorescence microscopy or flow cytometry (BD Biosciences, Accuri) as described above and in Lu et al., Nucleic Acids Res. 2019 doi: 10.1093 / nar / gkz093. The analysis was performed using C6).

[0247] AAV6 virus production and transduction: Adeno-associated viruses expressing SaCas9 and HBB sgRNA1 were generated from AAV vectors pSaCas9 (expressing SaCas9) and pSaCas9-HBB-sgRNA1 (expressing HBB sgRNA1 and containing a donor template for homologous recombination that modifies the wild-type HBB gene with a Sickle mutation), respectively. AAV serotype 6 (AAV6) production and quantification were performed by Virovek, Inc. (Hayward, CA). AAV6 transduction was performed in serum-free medium or OPTI-MEM with a viral genome of 10 3 ~10 4 The procedure was performed using cell titers. After 24 hours of transduction, the cells were returned to serum-containing growth medium.

[0248] The lentiviral vector plasmid pCK002-HBB-sgRNA1, expressing both SaCas9 and HBB sgRNA1, was used to produce integrated competent lentiviral vectors (packaged by packaging plasmid pspAX2) and integrated defective lentiviral (IDLV) vectors (packaged by packaging plasmid pspAX2-D64V) as described above. SaCas9 mRNA LVLP production was also described above. To produce Cas9 / sgRNA RNP LVLP, 13 million actively dividing HEK293T cells grown in a 15cm dish were converted to 10ml Opti-MEM. 16 μg of ABP-modified packaging plasmid pspAX2-D64V-NC-ABP (ABP may be MCP, PCP, λ N22, or COM), 6 μg of envelope plasmid (pMD2.G), and 16 μg of plasmid DNA co-expressing SaCas9 and aptamer-modified sgRNA were mixed in 1 ml of Opti-MEM. 76 μl of 1 mg / ml polyethyleneimine (PEI, Polysciences Inc.) was mixed in 1 ml of Opti-MEM. The DNA mixture and PEI mixture were then mixed and incubated at room temperature for 15 minutes. The DNA / PEI mixture was then added to the cells in Opti-MEM. 24 hours after transfection, the medium was changed to 15 ml of Opti-MEM, and Cas9 / sgRNA RNP LVLP was collected twice, once every 24 hours. The supernatant was rotated at 500 g for 10 minutes to remove cell debris before further processing as described below.

[0249] Lentivirus and LVLP concentration: The recovered and clarified supernatant was concentrated using the KR2i TFF System (KrosFlo® Research 2i Tangential Flow Filtration System) (Spectrum Lab, catalog no. SYR2-U20) in concentrate-diafiltration-concentrate mode. Typically, 150–300 ml of supernatant was first concentrated to approximately 50 ml, diafiltration with 500 ml to 1000 ml of PBS, and finally concentrated to approximately 8 ml. Hollow fiber filter modules were prepared from modified polyethersulfone with a molecular weight cutoff of 500 kDa. Flow rate and pressure limits were 80 ml / min and 8 psi for filter module D02-E500-05-N, and 10 ml / min and 5 psi for filter module C02-E500-05-N.

[0250] Lentiviral vector and LVLP quantification: Viral titers were determined by p24-based ELISA (Cell Biolabs, QuickTiter® Lentivirus Titer Kit, catalog number VPK-107). When assaying unpurified samples, viral particles were precipitated according to the manufacturer's instructions to ensure that soluble p24 protein was not detected.

[0251] Western blot analysis of viral proteins from lentiviruses and LVLPs. Purified lentiviruses or LVLPs (200 ng p24 by ELISA) were dissolved in 20 μl of 1× Laemmli sample buffer. Proteins in each sample were separated by SDS-PAGE gel and analyzed by Western blotting. Antibodies used included mouse monoclonal anti-SaCas9 antibody (Millipore Sigma, MAB131872, clone 6F7, 1:1000), rabbit polyclonal HIV1 p17 antibody against MA (ThermoFisher Scientific, catalog number PA1-4954, 1:1000), rabbit polyclonal HIV1 p15 antibody against NC (Abcam, catalog number ab66951, 1:1000), and p24 mouse monoclonal antibody against CA (Cell Biolabs, catalog number 310810, 1:1000). HRP-conjugated anti-mouse IgG(H+L) (ThermoFisher Scientific, catalog number 31430, 1:5000) and anti-rabbit IgG(H+L) (catalog number 31460, 1:5000) secondary antibodies were used in Western blotting. Chemiluminescent reagents (Pierce) were used to visualize protein signals under the LAS-3000 system (Fujifilm).

[0252] RNA Isolation and RT-qPCR Analysis from Lentiviruses or LVLPs: The miRNeasy Mini Kit (QIAGEN catalog number 217004) was used to isolate RNA from enriched lentiviruses or LVLPs. QuantiTect The Reverse Transcription Kit (QIAGEN) was used to reverse transcribe RNA into cDNA. For sgRNA reverse transcription, the half-random primer and half-sgRNA-specific primer (sgRNA-R2) provided in the kit were used. Custom-designed hydrolysis probes specific to SaCas9 and EGFP (ThermoFisher Scientific) were used with TaqMan Universal PCR Master. It was used in qPCR with Mix (ThermoFisher Scientific). HBB sgRNA1 and HBB sgRNA1 テトラ-com For detection, sgRNA-F1 (SEQ ID NO: 30) and s0067RNA-R3 were used as primers in SybrGreen-based RT-qPCR. PCR was performed using an ABI7500 instrument.

[0253] Membrane removal from LVLP core capsids: Lentivirus-like particles (Wiegers et al.) The LVLPs were transiently treated with 0.5% Triton® X-100 as described in al. J. Virol. 1998, 72: 2846-2854. Briefly, the LVLPs were centrifuged using a Sorvall T-890 rotor (2 hours, 120,000 g) through a step gradient containing a 1 ml layer of 10% sucrose in STE [100 mM NaCl, 50 mM Tris / HCl (pH 7.5), 1 mM EDTA] with or without 0.5% Triton® X-100, and a cushion of 2 ml of 20% sucrose in the STE solution. The pelletized viruses were directly lysed for Western blotting or RT-qPCR analysis.

[0254] Lentivirus and LVLP transduction: Cells grown in 24-well plates containing 8 μg / ml polyblen with varying amounts of concentrated lentivirus or LVLP (equivalent to 10-300 ng p24 protein) were measured in 2.5 × 10⁶ cells. 4The virus was added to each cell. The unconcentrated virus-containing supernatant was diluted in a 1:1 ratio with fresh medium for transduction into the cells. The cells were incubated in particle-containing medium for 12-24 hours, then replaced with standard medium.

[0255] For gene editing in human cells using Cas9 expressed from AAV serotype 6, SaCas9 expressing AAV6 and HBB sgRNA1 expressing AAV6 were simultaneously transduced into GFP reporter cells. For gene editing using LV or IDLV (packaged using a packaging plasmid containing a D64V mutation in integrase) expressing both SaCas9 and HBB sgRNA1, 10-300 ng of virus equivalent to p24 was introduced into 24-well plates in 2.5 × 10⁶ cells. 4 These methods were used for individual transduction. For gene editing using SaCas9 mRNA LVLP, various amounts of SaCas9 mRNA LVLP (quantified by p24) were simultaneously transduced into HEK293T cells or GFP reporter cells using IDLV expressing HBB sgRNA1. For transduction using Cas9 / sgRNA RNP LVLP, various amounts of Cas9 / sgRNA RNP LVLP were transduced into human cells. After 48-72 hours of transduction, gene editing activity was analyzed by GFP reporter assay or next-generation sequencing.

[0256] To investigate gene editing in human lymphoblastoid cells immortalized by Epstein-Barr virus transformation, human lymphoblastoid cell lines with and without the sickle cell mutation were purchased from the Corrie Institute (GM16265, with the sickle cell mutation; ID00085, with a mutation in the IL2RG gene). Lymphoblasts were cultured in RPMI 1640 containing 2 mmol / L L-glutamine and 15% fetal bovine serum at 37°C under 5% carbon dioxide. For LVLP and IDLV transduction, 2 × 10⁶ cells were used. 5Each cell was added to 0.5 ml of RPMI growth medium. Cas9 / sgRNA RNP LVLP was then added to the cells. Polybren was added to the medium at a final concentration of 8 μg / ml. The cells were replaced with fresh medium 20 hours after transduction. The cells were harvested 72 hours after transduction for DNA analysis by next-generation sequencing.

[0257] Next-generation sequencing and data analysis: Endogenous HBB target sequences and IL2RG target sequences in the embedded GFP expression cassette, as well as the HBB and IL2RG target sequences, were amplified for sequencing analysis. A nested PCR strategy was used to amplify the endogenous HBB target sequence to avoid amplifying sequences derived from the viral vector template. First, primers HBB-1849F and HBB-5277R were used to amplify the 3.4kb region derived from the HBB gene locus. These two primers cannot amplify sequences derived from the template in the viral vector. Next, HBB-F1 and HBB-F2 primers were used to amplify the target DNA for sequencing. To amplify the endogenous IL2RG target sequence, primers IL2RG-1029F and IL2RG-3301R were used to amplify the target region from cells treated with these primers (it was not possible to amplify the template-derived sequence in the viral vector). Subsequently, primers IL2RG-F1 (SEQ ID NO: 90) and IL2RG-3301R were used to amplify the target DNA from the first PCR product for sequencing. For sequencing, reporter F and reporter R1 primers were used to amplify the HBB target sequence derived from the integrated EGFP reporter. Proofreading HotStart® ReadyMix from KAPA Biosystems (Wilmington, MA) was used for PCR. The purified PCR products were sent to Genewiz Inc. (Morrisville, NC) for next-generation sequencing (Amplicon EZ). Typically, 50,000 read data points per amplicon were obtained.

[0258] After removing the 3' linker and 5' barcode sequences, the obtained read data was sent to online Cas-Analyzer software for mutation analysis. When sending the reference sequence, primer sequences were excluded if they were at least 12nt away from the sgRNA target. Otherwise, primer sequences were included in the reference. Only read data containing both 5'12nt and 3'12nt of the reference sequence (excluding primer sequences) were included in the indel analysis. The total indel rate was the difference between 1 and the percentage of read data that did not contain mutations. The top 8-10 most frequently observed read data are shown.

[0259] Monitoring the rate of GFP-positive cell emergence: 2.5 × 10⁶ GFP reporter cells seeded in a 24-well plate. 4 Each individual contains 50ng p24 Cas9 / IL2RG sgRNA1 テトラ-com Cells were transduced using RNP LVLPs, or co-transduced using 100 ng p24 IDLVs expressing Cas9 mRNA LVLP and IL2RG sgRNA1. Cells were then incubated in an IncuCyte S3 system (Essen BioScience, Inc., Ann Arbor, Michigan) for timed GFP fluorescence scanning. Two wells were scanned from each treatment, and nine spots were scanned from each well. Scanning began immediately after transduction, and cells were scanned once every two hours for 48 hours. The GFP positivity rate for each image was calculated by dividing the GFP-positive area in the image by the phase area (area occupied by cells).

[0260] Statistical analysis was performed using GraphPad Prism software. A t-test was used to compare the means of two groups. For data from more than two groups, analysis of variance (ANOVA) was performed, followed by Tukey's post-hoc test. For two-factor analyses, Bonferroni post-hoc test was performed after ANOVA. A p-value of <0.05 was considered statistically significant.

[0261] Replacing the sgRNA tetraloop with an aptamer best preserved Cas9 / sgRNA RNP activity. We examined the packaging of Cas9 / sgRNA RNP into lentivirus-like particles. The overall strategy is to incorporate ABP into lentivirus-like particles by fusing ABP to the lentivirus nucleocapsid protein (NC) as described above, adding the corresponding aptamer that complexes with the Cas9 protein to the sgRNA, forming the Cas9 / sgRNA RNP during lentivirus capsid assembly. The Cas9 / sgRNA RNP is packaged to the lentivirus capsid via a specific aptamer / ABP interaction (Figure 14A, right). After evasion from endosomes, the Cas9 / sgRNA RNP is released into the cytoplasm following capsid decoating, and the RNP complex then enters the nucleus to perform gene editing.

[0262] To implement this strategy, it was necessary to find a location within the sgRNA scaffold that best accommodates aptamer insertion and preserves nuclease activity after complexing with Cas9. The MS2 aptamer was used because it mediates efficient Cas9 mRNA packaging. Three locations were examined: MS2 insertion into stem-loop 2 (ST2), replacement of the tetraloop with MS2, and addition of MS2 after the 3' end of the sgRNA (Figure 14B). When plasmid DNA co-expressing SaCas9 and a modified sgRNA targeting HBB sgRNA1 was transfected into GFP reporter cells, indels in the HBB sgRNA1 target sequence could restore GFP expression. Replacing the tetraloop or ST2 loop slightly reduced the percentage of GFP-positive reporter cells, but adding one MS2 to any of the three locations was found to preserve gRNA activity in transfection experiments (Figure 14C). Addition of two MS2 aptamers was also examined. One aptamer replaced the tetraloop, while the other was located in the ST2 loop or the 3' end. In both cases, the GFP-positive percentage consistently decreased, consistent with the observation that more than one copy of MS2 reduces RNA stability. Therefore, we used one copy of the aptamer in further experiments.

[0263] We also examined whether these MS2-modified sgRNAs could be packaged and delivered by LVLP. SaCas9 protein and MS2-modified HBB sgRNA1 were co-expressed during LVLP preparation, and the LVLPs were used to transduce our GFP reporter cells. Flow cytometry analysis revealed that LVLPs containing HBB sgRNA1 with MS2 in a tetraloop (HBB sgRNA1) were... テトラ MS2 ) yields the most GFP-positive cells, and LVLP (HBB sgRNA1) contains MS2 at its 3' end. 3’MS2 ) followed this, and it was found that MS2 (HBB sgRNA1) in stem-loop II ST2 MS2In contrast to the transfection experiments in which ) was active, HBB sgRNA1 ST2 MS2 LVLP has virtually no gene editing activity (Figure 14D), and HBB sgRNA1 ST2 MS2 This suggests that it is either unpackable or does not leave any post-transformation processes.

[0264] com / COM is an aptamer / ABP pair for sgRNA packaging. Given that replacing the tetraloop with an aptamer proved to be the best approach, additional aptamers that could be used to replace the tetraloop were investigated. Four aptamers, MS2(23), PP7(24), BoxB(25), and com(26), were used in sgRNA for target binding purposes (22, 27-29). The activity of HBB sgRNA1 containing various aptamers (MS2, PP7, BoxB, and com) replacing the tetraloop was examined (Figure 15A). SaCas9 and various aptamer-modified HBBs were also examined. When plasmid DNA co-expressing sgRNA1 was transfected into GFP reporter cells, replacing the tetraloop with the com aptamer produced the same rate of GFP-positive cells as with unmodified sgRNA, while replacing the tetraloop with the other three aptamers significantly reduced the GFP-positive rate (Figure 15B).

[0265] We examined the packaging of HBB sgRNA1 to LVLPs containing different aptamers. For this purpose, we used modified packaging plasmids of MCP (MS2 coat protein, binding to MS2), PCP (PP7 coat protein, binding to PP7), λN22 peptide (binding to BoxB), and COM (binding to com), in which ABP was inserted after the second zinc finger domain of the nucleocapsid protein (NC) as described above, and Cas9 was used to conjugate them with various modified HBB sgRNA1s to produce RNP-containing LVLPs. These LVLPs were transduced into GFP reporter cells. Flow cytometry found that LVLPs produced by the com / COM pair had the highest number of GFP-positive cells, followed by those produced by MS2 / MCP. PP7 / PCP and BoxB / λN22 produced LVLPs with the lowest activity (Figure 15C). GFP-positive cells were observed only when these LVLPs were used to transduce GFP reporter cells, but were not found in HEK293T cells, ruling out the possibility of contamination with GFP-expressing DNA or viruses (Figure 16).

[0266] Since the com-COM combination yielded the best results, we investigated whether COM modification of the packaging plasmid impairs particle assembly. A slight decrease of only 11% in particle assembly efficiency was observed compared to the unmodified packaging plasmid (Figure 17). This slight decrease should not prevent sufficient particle production. Through these experiments, we identified the most efficient packaging site (tetraloop) and aptamer / ABP pair (com / COM) for Cas9 RNP (or Cas9 mRNA and sgRNA) to the lentiviral capsid.

[0267] Cas9 / sgRNA RNPs in LVLPs were the primary cause of the observed gene editing activity. While designed to package Cas9 / sgRNA RNPs, considering previous observations that aptamer-free SaCas9 mRNA may be packaged by an unknown mechanism, the observed gene editing activity may originate from co-packaged SaCas9 mRNA and sgRNA. (Cas9 / sgRNA RNP or SaCas9) To determine whether mRNA / sgRNA contributes to observed gene editing activity, Cas9 expression cassettes were modified to HHB sgRNA1 テトラ-com The expression cassette was separated into two different plasmids. When the two plasmids were co-transfected into GFP reporter cells, they generated GFP-positive cells with the same efficiency as when a single plasmid containing both expression cassettes was transfected (Figure 18A). However, in the absence of Cas9 expression, HHB sgRNA1 テトラ-com When packaged in LVLPs, these LVLPs exhibited little to no gene editing activity when co-transduced into GFP reporter cells using validated functional Cas9 mRNA LVLPs (Figure 18B).

[0268] The Cas9 protein is required to protect the stability of sgRNA in cells. This is HBB sgRNA1 テトラ-com This was confirmed by expressing Cas9 alone or by co-expressing Cas9 with sgRNA. Co-expression of Cas9 was confirmed by HBB sgRNA1 テトラ-com The expression of was significantly increased (Figure 18C). After DNA transfection, sgRNA was constitutively expressed. On the other hand, after transduction, sgRNAs completed posttransduction intracellular transport, and no new sgRNAs were generated. Therefore, the protective effect of the Cas9 protein on sgRNA is predicted to be even more important. These data suggest that sgRNA instability, particularly the lack of ability of sgRNAs to survive posttransduction intracellular transport, is related to single-packaged HHB sgRNA1. テトラ-comThis suggests that it primarily explains why LVLPs are inactive after co-transduction with Cas9 mRNA LVLPs.

[0269] These experiments demonstrate that, in addition to co-packaged Cas9 mRNA and sgRNA, packaged Cas9 / sgRNA RNPs can also be used for gene editing. HHB sgRNA1 packaged with Cas9 expression テトラ-com Western blot analysis of enriched LVLPs was performed to examine the presence of Cas9 protein in LVLPs (Figure 18D). Viral proteins MA (p17) and CA (p24) were detected at the predicted size and quantity, indicating that the processing of MA and CA was unaffected. The detection of p15, which is then processed by NC, showed a band between 10kd and 15kd in GFP lentivirus (lane 1) and in LVLPs generated with the unmodified packaging plasmid (lane 3). In LVLPs generated with the NC-MCP modified packaging plasmid (lane 2), a strong band was detected between 15 and 20kd, which was slightly smaller than the predicted 21.8kd NC-MCP fusion protein. However, in LVLPs generated with the NC-COM modified packaging plasmid (lanes 4-6), a band slightly smaller than 15kd was detected, which was slightly smaller than the predicted 18.7kd NC-COM fusion protein. Anti-p15 bands were detected in all samples (including GFP lentivirus) that were slightly smaller than expected, potentially resulting from the SDS-PAGE system or partial degradation of p15 or p15 fusion proteins.

[0270] Surprisingly, the Cas9 protein was detected in all types of LVLPs produced in Cas9-expressing cells, including samples that did not contain sgRNA (lane 3, observed after long-term exposure) or samples that did not contain the tetra-com aptamer in the sgRNA (lane 6). However, Cas9 was not detected in GFP-expressing lentiviruses (Figure 18D, lane 1). The detection of the Cas9 protein in LVLPs suggests that the Cas9 protein can contribute to the observed gene editing activity, but cannot explain the lack of activity from LVLPs containing sgRNA without the Cas9 protein and com modification.

[0271] The com modification of sgRNA was necessary for efficient packaging of Cas9 / sgRNA RNPs into the core capsid of LVLPs. Cas9 protein was detected in LVLPs containing HBB sgRNA1 without the com modification (Figure 18D, lane 6). These showed minimal gene editing activity in a GFP reporter assay (Figure 18B). Therefore, the relative levels of Cas9 protein (normalized to capsid protein CA) in LVLPs with and without the com aptamer were compared to determine whether the amount of Cas9 protein per particle explains the difference in activity. Cas9 / HBB sgRNA1 テトラ-com LVLP(com + It was found that ) contains 2.9 times more Cas9 protein than Cas9 / HBB sgRNA1 LVLP (Figure 18E, com - ).

[0272] Since there was a difference of less than threefold in Cas9 protein content, we investigated the compartmentalization of Cas9 protein. Because LVLPs were enriched by tangential flow filtration, it was suggested that LVLPs may possess membrane structures containing exosomes. It was hypothesized that RNPs within the capsid core are most likely to conserve gene editing activity during intracellular transport after transduction, and therefore the amount of surfactant-resistant Cas9 protein is important. To detect Cas9 protein within the capsid core, particles were transiently treated with 0.5% Triton® X-100 to remove Cas9 protein associated with membrane vesicles and the capsid envelope. Triton® X-100 treatment significantly reduced the amount of MA protein associated with the capsid envelope (detected by p17 antibody), indicating that the treatment was effective (Figure 18F). CA protein was reduced to different degrees, which may reflect different core-capsid stabilities in different types of particles. However, Cas9 protein levels were significantly reduced in LVLPs with unmodified sgRNA, but only slightly reduced in LVLPs with tetra-com modified sgRNA. Cas9 / HBB sgRNA1 テトラ-com LVLPs possessed 6.8 times more surfactant-resistant Cas9 protein compared to Cas9 / HBB sgRNA1 LVLPs. These data suggest that tetra-com modification of sgRNA promotes the packaging of Cas9 protein in surfactant-resistant capsid cores, and that the amount of core-protective Cas9 / sgRNA RNPs correlates with gene editing activity.

[0273] The importance of com-aptamers in sgRNA packaging was investigated by RT-qPCR analysis of sgRNA content in LVLPs. The PCR primers used were HBB sgRNA1 and HBB sgRNA1 テトラ-comAlthough amplicons of different sizes were produced, the primers produced very similar standard curves when different amounts of plasmid DNA were used as templates for qPCR due to the presence and absence of the 23nt com-aptamer (Figure 19), demonstrating similar amplification efficiencies for com-positive and com-negative sgRNA sequences. In LVLP without Cas9 protein, HHB sgRNA1 テトラ-com The levels were 2.5 times higher in LVLPs containing Cas9 protein. However, in LVLPs containing Cas9 and com-negative HBB sgRNA1, the sgRNA levels were higher in Cas9 and HBB sgRNA1. テトラ-com The amount was less than 1 / 50 of that in LVLPs containing (Figure 18G). These data indicated that sgRNA packaging is com-aptamer dependent but not Cas9 protein dependent. The Cas9 protein slightly reduced the amount of packaged com-modified sgRNA. This is likely due to the negative effect of Cas9 / sgRNA association on com / COM interaction. Therefore, these data suggest that Cas9 protein packaging to the capsid core is dependent on sgRNA via com / COM interaction. テトラ-com It was shown that this is mediated by the Cas9 protein. The Cas9 protein is not required for sgRNA packaging to LVLP, but it is required to protect sgRNA during transduction. Except for the experiment shown in Figure 18, where sgRNA with and without the com modification were used for comparison, com-modified sgRNA was used in all subsequent experiments. Hereafter, for brevity, Cas9 / sgRNA RNPs are used instead of Cas9 / sgRNA RNPs. テトラ-com Used to demonstrate RNP

[0274] Cas9 / sgRNA RNP LVLP enabled efficient genome editing and improved off-target identification. The gene editing activity of the described Cas9 RNP LVLP and the Cas9 mRNA LVLP described above was compared. Cas9 / HHB sgRNA1 RNP LVLP showed comparable gene editing activity to SaCas9 mRNA LVLP in a GFP reporter assay (Figure 20A).

[0275] The gene-editing activity of RNP LVLP was further investigated by next-generation sequencing (NGS) using the IL2RG sgRNA1 described above for IL2RG, another target whose mutations cause X-linked severe combined immunodeficiency. GFP reporter cells 2.5 × 10⁶ were treated with Cas9 / IL2RG sgRNA1 RNP LVLP. 4 Transduction was performed on individual cells. 72 hours after transduction, the endogenous IL2RG region was amplified and subjected to NGS. These particles of 15, 45, and 100 ng p24 produced 4.1%, 8.1%, and 21.1% indels in the endogenous IL2RG gene (Figure 20B). In the studies described above, 30 ng p24 SaCas9 mRNA LVLP and 60 ng IL2RG sgRNA LVLP produced 13% indels in the IL2RG of HEK293T cells; therefore, these RNP LVLPs showed comparable or even better gene editing activity. 200 ng p24 IL2RG sgRNA1 RNP LVLP was used on B lymphoblast-like cells 2 × 10⁶ 5 Transduction was performed on individual cells, and 18.3% indels were detected (Figure 21). This activity is comparable to that of 100ng p24 SaCas9 mRNA LVLP and 100ng p24 IL2RG sgRNA1-expressing IDLV, which produced 11% indels in the same number of B lymphoblast-like cells.

[0276] These data showed that RNP-LVLP was as active in gene editing as Cas9 mRNA-LVLP. RNP-mediated Cas9 delivery should provide better regulation of the amount of Cas9 protein delivered per cell and further transient Cas9 action. Both of these would help reduce off-target rates. All nine predicted potential HBB sgRNA1 off-targets have very low indels, hindering a comparison of off-target rates between different delivery methods. However, the wild-type HBB sequence corresponding to the sickle cell mutation has only one nucleotide mismatch in HBB sgRNA1 (Figure 20C), and a detectable off-target indel could be generated by Cas9 / HBB sgRNA1. Therefore, in GFP reporter cells, we studied the on-target indel rate (in sickle cell mutations in GFP reporter cassettes, shown below in Figure 20C) and off-target indel rate (endogenous wild-type HBB sequence with a one-nucleotide mismatch to HBB sgRNA1, shown above in Figure 20C) when Cas9 / HBB sgRNA1 was delivered by plasmid transfection or by LV, IDLV, and AAV. The results showed that Cas9 / HBB sgRNA1 RNP LVLP had the highest ratio of on-target indel rate to off-target indel rate, and Cas9 mRNA / HBB sgRNA1 LVLP had the second highest, both higher than those of LV, IDLV, and AAV (Figure 20D). Thus, RNP LVLP best distinguished on-target effects from off-target effects.

[0277] Cas9 / sgRNA RNP LVLP showed a faster effect than Cas9 mRNA LVLP after transduction. The GFP reporter assay was performed using Cas9 / sgRNA RNP. This was used to compare the dynamics of GFP-positive cell emergence after LVLP and Cas9 mRNA LVLP treatment. For this purpose, 2.5 × 10⁶ GFP reporter cells were used. 4Cells were transduced using 50 ng p24 Cas9 / IL2RG sgRNA1 RNP LVLP, or GFP reporter cells were co-transduced using 100 ng p24 Cas9 mRNA LVLP and 100 ng p24 IDLV expressing IL2RG sgRNA1. The appearance of GFP-positive cells was monitored every 2 hours. GFP-positive cells appeared at least 6 hours earlier in RNP LVLP-treated cells than in mRNA LVLP-treated cells (Figure 20E). The GFP-positive area percentages for the two treatments converged 34 hours after transduction. The data indicated that mRNA LVLP may be effective, but RNP LVLP has a faster effect than mRNA LVLP and IDLV. This is thought to be because RNP is available for nuclear function immediately after escape from endosomes, while mRNA LVLP and IDLV require more time to express Cas9 protein and sgRNA. The differences in dynamics also indicate that the functional components in Cas9 / sgRNA RNP LVLPs differ from those in Cas9 mRNA LVLPs.

[0278] LVLPs mediate efficient homologous recombination in the presence of a donor template. One application of CRISPR / Cas9 is to promote homologous recombination in the presence of a donor template. Integrated defect lentiviral vectors were used to prepare the donor template. COM modification of NC in the packaging plasmid slightly reduced packaging of GFP-expressing lentiviral vectors (1.0±0.15 for unmodified packaging plasmids, N=3; 0.73±0.15 for NC-COM modified packaging plasmids, N=6; p>0.05), but almost completely removed GFP expression after transduction of these COM-modified GFP lentiviral vectors. In addition, packaging of Cas9 / sgRNA RNP reduced Ψ-mediated lentiviral genome packaging, but the difference did not reach statistical significance (with RNP, 0.53±0.16, N=3; without RNP, 0.93±0.21, N=3; p>0.05). These observations suggest that the donor templates were delivered separately by embedded defective lentiviral vectors (IDLVs).

[0279] Lentiviral vectors are widely used for ex vivo modification of cells. LVLP was used to facilitate the insertion of functional IL2RG cDNA downstream of the endogenous IL2RG promoter. This cDNA insertion strategy can be used to treat SCID-X1 regardless of the nature of the disease-causing mutation. The donor template contains a 1.5kb 5' homologous arm, a 1.1kb IL2RG cDNA, and a 3' homologous arm. The 3' homologous arm was designed so that the cDNA replaces a similar length of IL2RG genomic DNA after homologous recombination. The IL2RG cDNA was codon-optimized to maximize the difference from the original cDNA to reduce the chance of cDNA-mediated homologous recombination. In addition to the donor template, the lentiviral vector also contained two U6 promoters driving the expression of IL2RG sgRNA1 and sgRNA2 to facilitate cDNA insertion (Figure 22A).

[0280] Donor template / sgRNA was delivered to lymphoblastic cells using IDLV containing or not containing Cas9 / IL2RG sgRNA1 RNP LVLP. 72 hours after transduction, genomic DNA was extracted and the target region was amplified by two rounds of PCR amplification (Figure 22A). In the first PCR, one primer was outside the donor template and therefore could not be amplified from the template DNA without homologous recombination. The product of the first PCR was purified from the gel and used as a template in the second PCR to amplify the 5' and 3' junction DNA for sequencing. Three primers were used in each PCR in addition to the common primer. One primer matched the cDNA sequence inserted to amplify the HR-containing DNA, and another primer matched the genomic DNA that was replaced to amplify the HR-free DNA. HEK293T cells 2.5 × 10⁶ were subjected to LVLP and IDLV with 100 ng p24. 4 Transduction was performed on individual cells. At the 5' junction, 46.2% of the sequences were observed to be HR-free (29.6% were indel-free, and the remaining 16.6% were indel-free), and 53.8% of the sequences were HR-free (Figure 22B). In the read data containing HR, the indel rate was similar to the background, as the target sequence was not present in the donor template. The data indicated that Cas9 / sgRNA RNP was active and significantly enhanced targeted insertions of 1.1kb DNA. The observation that 57% of the read data in the PCR-produced cells contained HR suggests that these LVLPs may be used in conjunction with IDLV to enhance HDR. The present invention provides, for example, the following items. (Item 1) A lentiviral packaging plasmid comprising a eukaryotic cell promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, and one or both of the NC coding sequence or the MA coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence, and the packaging plasmid does not encode a functional integrase protein. (Item 2) The lentiviral packaging plasmid described in item 1, wherein the NC coding sequence comprises two functional zinc finger protein domains and a functional native protease processing sequence. (Item 3) The lentiviral packaging plasmid according to item 1 or 2, wherein at least one nonviral ABP nucleotide sequence encodes an MS2 coat protein, a PP7 coat protein, a lambda N peptide, or a COM protein. (Item 4) A lentiviral packaging plasmid according to any one of items 1 to 3, wherein the Gag nucleotide sequence comprises a first nonviral ABP nucleotide sequence and a second nonviral ABP nucleotide sequence in tandem. (Item 5) The lentiviral packaging plasmid according to item 4, wherein both the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence encode the same ABP, and the ABP comprises an MS2 coat protein, a PP7 coat protein, a lambda N peptide, or a COM protein. (Item 6) The lentiviral packaging plasmid described in item 4, wherein the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence encode different ABPs selected from the group consisting of MS2 coated protein, PP7 coated protein, lambda N peptide, and COM protein. (Item 7) The lentiviral packaging plasmid according to any one of items 1 to 6, wherein the NC coding sequence comprises at least one first nonviral ABP nucleotide sequence, and the MA coding sequence comprises at least one second nonviral ABP nucleotide sequence. (Item 8) The lentiviral packaging plasmid according to item 7, wherein both the at least one first nonviral ABP nucleotide sequence and the at least one second nonviral ABP nucleotide sequence encode the same ABP, and the ABP comprises an MS2 coat protein, a PP7 coat protein, a lambda N peptide, or a COM protein. (Item 9) A lentiviral packaging plasmid according to item 7 or 8, wherein the at least one first nonviral ABP nucleotide sequence and the at least one second nonviral ABP nucleotide sequence encode different ABPs selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N peptide, and COM protein. (Item 10) A lentiviral packaging plasmid according to any one of items 1 to 9, further comprising a Rev nucleotide sequence and a Tat nucleotide sequence. (Item 11) A lentiviral packaging plasmid as described in any one of items 1 to 10, comprising an integrase coding sequence having an integrase inactivation mutation. (Item 12) The lentiviral packaging plasmid described in item 11, wherein the integrase inactivation mutation is a mutation from aspartic acid to valine (D64V) at amino acid position 64 of the integrase protein encoded by the integrase coding sequence. (Item 13) A lentiviral packaging plasmid as described in any one of items 1 through 12, comprising the deletion of all or part of the integrase coding sequence. (Item 14) A lentiviral packaging plasmid as described in item 1, containing all or part of the reverse transcriptase coding sequence. (Item 15) The lentiviral packaging plasmid according to any one of items 1 to 14, wherein the eukaryotic cell promoter is an RNA polymerase II promoter. (Item 16) A mammalian expression plasmid comprising a eukaryotic cell promoter operably linked to a viral protein R (VPR) coding sequence or a negative regulator (NEF) coding sequence, wherein the VPR coding sequence or the NEF coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence. (Item 17) The mammalian expression plasmid described in item 16, wherein at least one nonviral ABP nucleotide sequence encodes an MS2 coat protein, a PP7 coat protein, a lambda N peptide, or a COM protein. (Item 18) A mammalian expression plasmid according to item 16 or item 17, wherein the VPR coding sequence or the NEF coding sequence comprises a first nonviral ABP nucleotide sequence and a second nonviral ABP nucleotide sequence. (Item 19) The mammalian expression plasmid described in item 18, wherein the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence are the same nonviral ABP nucleotide sequence. (Item 20) The mammalian expression plasmid described in item 18, wherein the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence are different nonviral ABP nucleotide sequences. (Item 21) The mammalian expression plasmid described in item 19, wherein both the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence encode an ABP selected from the group consisting of MS2 coated protein, PP7 coated protein, lambda N peptide, and COM protein. (Item 22) The mammalian expression plasmid described in item 20, wherein the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence each encode a different ABP selected from the group consisting of MS2 coated protein, PP7 coated protein, lambda N peptide, and COM protein. (Item 23) The lentiviral packaging plasmid according to any one of items 16 to 22, wherein the eukaryotic cell promoter is an RNA polymerase II promoter. (Item 24) A mammalian expression plasmid comprising a eukaryotic cell promoter operably ligated to a nonviral nucleic acid sequence, wherein the nonviral nucleic acid sequence comprises at least one aptamer coding sequence, and the nonviral nucleic acid sequence comprises (i) one or both of a CRISPR-related endonuclease coding sequence or a guide RNA (gRNA) coding sequence and (ii) at least one aptamer coding sequence. (Item 25) The mammalian expression plasmid described in item 24, wherein the CRISPR-related endonuclease coding sequence encodes the Cas9 protein, the Cpf1 protein, or a derivative thereof. (Item 26) A mammalian expression plasmid according to item 24 or 25, wherein the gRNA coding sequence encodes an RNA molecule comprising a DNA targeting sequence and a constant region that interacts with the CRISPR-associated endonuclease. (Item 27) A mammalian expression plasmid according to any one of items 24 to 26, wherein the gRNA coding sequence codes for a gRNA containing a transactivating crRNA (tracrRNA) sequence. (Item 28) A mammalian expression plasmid according to any one of items 24 to 27, wherein the gRNA coding sequence codes for a gRNA that does not contain a tracrRNA sequence. (Item 29) The mammalian expression plasmid according to any one of items 24 to 28, wherein the nonviral nucleic acid sequence is a CRISPR-related endonuclease coding sequence, and the eukaryotic cell promoter operably ligated thereto is an RNA polymerase II promoter. (Item 30) The mammalian expression plasmid according to any one of items 24 to 28, wherein the nonviral nucleic acid sequence is a gRNA coding sequence, and the eukaryotic cell promoter operably ligated thereto is an RNA polymerase III promoter. (Item 31) The mammalian expression plasmid according to any one of items 24 to 28, wherein the nonviral nucleic acid sequence comprises both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, the RNA polymerase II promoter is operably ligated to the CRISPR-associated endonuclease coding sequence, and the RNA polymerase III promoter is operably ligated to the gRNA coding sequence. (Item 32) The mammalian expression plasmid according to any one of items 24 to 31, wherein the at least one aptamer coding sequence codes for an aptamer sequence to which ABP is specifically bound, selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or COM protein. (Item 33) A mammalian expression plasmid according to any one of items 24 to 32, wherein the nonviral nucleic acid sequence comprises two aptamer coding sequences. (Item 34) A mammalian expression plasmid according to any one of items 24 to 33, wherein at least one of the nonviral nucleic acid sequences comprises a CRISPR-associated endonuclease coding sequence including at least one first aptamer coding sequence and / or a gRNA coding sequence including at least one second aptamer coding sequence. (Item 35) A mammalian expression plasmid according to any one of item 34, wherein at least one of the nonviral nucleic acid sequences comprises a CRISPR-related endonuclease coding sequence that includes at least one aptamer coding sequence. (Item 36) The mammalian expression plasmid described in item 34, wherein the gRNA coding sequence comprises at least one aptamer coding sequence. (Item 37) The mammalian expression plasmid according to item 36, wherein at least one aptamer coding sequence is inserted within the tetraloop of the gRNA coding sequence. (Item 38) The mammalian expression plasmid described in item 37, wherein the aptamer coding sequence binds to the COM protein. (Item 39) The mammalian expression plasmid according to item 34, wherein the at least one first aptamer coding sequence and the at least one second aptamer coding sequence are the same aptamer coding sequence. (Item 40) The mammalian expression plasmid according to item 34, wherein the at least one first aptamer coding sequence codes for an aptamer sequence to which a first ABP specifically binds, the at least one second aptamer coding sequence codes for an aptamer sequence to which a second ABP specifically binds, and the at least one first aptamer coding sequence and the at least one second aptamer coding sequence code for aptamer sequences to which different first and second ABPs bind. (Item 41) The mammalian expression plasmid according to item 40, wherein the at least one first aptamer coding sequence and the at least one second aptamer coding sequence code for an aptamer sequence to which ABP is specifically bound, selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA-binding domain, and COM protein. (Item 42) The mammalian expression plasmid according to item 40, wherein the at least one first aptamer coding sequence and the at least one second aptamer coding sequence each encode an aptamer sequence to which different ABPs selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA-binding domain, and COM protein are each specifically bound. (Item 43) A mammalian expression plasmid according to any one of items 24 to 42, wherein a polynucleotide sequence encoding an RNA-stabilizing sequence is located at the 3' end of the nonviral nucleic acid sequence. (Item 44) The mammalian expression plasmid described in item 43, wherein the polynucleotide sequence encoding the RNA stabilization sequence comprises a polynucleotide sequence encoding at least one 3'UTR of the human betaglobin gene. (Item 45) A mammalian expression plasmid as described in any one of items 24 to 44, wherein the plasmid is a lentiviral transfer plasmid. (Item 46) a) A packaging plasmid comprising a eukaryotic cell promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, and either or both of the NC coding sequence or the MA coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence, and the packaging plasmid does not encode a functional integrase protein, b) A mammalian expression plasmid comprising at least one eukaryotic cell promoter operably ligated to a nonviral nucleic acid sequence, wherein the nonviral nucleic acid sequence comprises a CRISPR-related endonuclease coding sequence, a guide RNA (gRNA) coding sequence, or both a CRISPR-related endonuclease coding sequence and a gRNA coding sequence, c) Envelope plasmid containing an envelope glycoprotein coding sequence and A lentivirus packaging system including [unclear]. (Item 47) A lentiviral packaging system as described in item 46, wherein the nonviral RNA sequence includes at least one aptamer sequence. (Item 48) The lentiviral packaging system according to item 46 or item 47, wherein the packaging plasmid further comprises a Rev nucleotide sequence and a Tat nucleotide sequence. (Item 49) The lentiviral packaging system described in item 46, further comprising a second packaging plasmid containing the Rev nucleotide sequence. (Item 50) The lentiviral packaging system according to any one of items 46 to 49, wherein the NC coding sequence comprises two functional zinc finger protein domains and a functional native protease processing sequence. (Item 51) The lentiviral packaging system according to any one of items 46 to 50, wherein the at least one nonviral ABP nucleotide sequence encodes an MS2 coat protein, a PP7 coat protein, a lambda N peptide, or a COM protein. (Item 52) A lentiviral packaging system according to any one of items 46 to 51, wherein one or both of the NC coding sequence or the MA coding sequence comprises a first nonviral ABP nucleotide sequence and a second nonviral ABP nucleotide sequence immediately downstream of the first nonviral ABP nucleotide sequence. (Item 53) The lentiviral packaging system according to item 52, wherein both the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence encode the same ABP, and the ABP comprises an MS2 coat protein, a PP7 coat protein, a lambda N peptide, or a COM protein. (Item 54) The lentiviral packaging system according to item 52, wherein the first nonviral ABP nucleotide sequence and the second nonviral ABP nucleotide sequence encode different ABPs selected from the group consisting of MS2 coated protein, PP7 coated protein, lambda N peptide, and COM protein. (Item 55) The lentiviral packaging system according to any one of items 46 to 54, wherein the NC coding sequence comprises a first nonviral ABP nucleotide sequence, and the MA coding sequence comprises at least one second nonviral ABP nucleotide sequence. (Item 56) The lentiviral packaging system according to item 55, wherein at least one of the first nonviral ABP nucleotide sequences and at least one of the second nonviral ABP nucleotide sequences both encode the same ABP, and the ABP comprises an MS2 coat protein, a PP7 coat protein, a lambda N peptide, or a COM protein. (Item 57) The lentiviral packaging system according to item 55, wherein at least one of the first nonviral ABP nucleotide sequences and at least one of the second nonviral ABP nucleotide sequences encode different ABPs selected from the group consisting of MS2 coated protein, PP7 coated protein, lambda N peptide, and COM protein. (Item 58) A lentiviral packaging system according to any one of items 46 to 58, wherein the lentiviral packaging plasmid comprises an integrase coding sequence having an integrase inactivating mutation. (Item 59) The lentiviral packaging system according to item 58, wherein the integrase inactivation mutation is a mutation from aspartic acid to valine (D64V) at amino acid position 64 of the integrase protein encoded by the integrase coding sequence. (Item 60) A lentiviral packaging system as described in any one of items 46 to 59, wherein the lentiviral packaging plasmid contains a deletion of all or part of the integrase coding sequence. (Item 61) A lentiviral packaging system as described in any one of items 46 to 60, wherein the lentiviral packaging plasmid contains a deletion of all or part of the reverse transcriptase coding sequence. (Item 62) The lentiviral packaging system according to any one of items 46 to 61, wherein the eukaryotic cell promoter operably linked to the Gag nucleotide sequence is an RNA polymerase II promoter. (Item 63) The lentiviral packaging system according to any one of items 46 to 62, wherein the CRISPR-related endonuclease coding sequence codes for the Cas9 protein, the Cpf1 protein, or a derivative thereof. (Item 64) The lentiviral packaging system according to any one of items 46 to 63, wherein the gRNA coding sequence codes for an RNA molecule comprising a DNA targeting sequence and a constant region that interacts with the CRISPR-associated endonuclease. (Item 65) A lentiviral packaging system according to any one of items 46 to 64, wherein the gRNA encoded by the gRNA coding sequence includes a transactivating crRNA (tracrRNA) sequence. (Item 66) A lentiviral packaging system according to any one of items 46 to 65, wherein the gRNA encoded by the gRNA coding sequence does not contain a tracrRNA sequence. (Item 67) The lentiviral packaging system according to any one of items 46 to 66, wherein the nonviral nucleic acid sequence is a CRISPR-associated endonuclease coding sequence, and the eukaryotic cell promoter operably linked thereto is an RNA polymerase II promoter. (Item 68) The lentiviral packaging system according to any one of items 46 to 67, wherein the nonviral nucleic acid sequence is a gRNA coding sequence, and the eukaryotic cell promoter operably ligated thereto is an RNA polymerase III promoter. (Item 69) The lentiviral packaging system according to any one of items 46 to 68, wherein the nonviral nucleic acid sequence comprises both a CRISPR-associated endonuclease coding sequence and a gRNA coding sequence, an RNA polymerase II promoter is operably ligated to the CRISPR-associated endonuclease coding sequence, and an RNA polymerase III promoter is operably ligated to the gRNA coding sequence. (Item 70) The lentiviral packaging system according to any one of items 47 to 69, wherein the at least one aptamer sequence encodes an ABP target RNA binding sequence for ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or COM protein. (Item 71) The lentiviral packaging system according to any one of items 47 to 70, wherein the nonviral nucleic acid sequence in the at least one mammalian expression plasmid comprises two aptamer sequences. (Item 72) A lentiviral packaging system according to any one of items 47 to 71, wherein at least one of the nonviral nucleic acids comprises a CRISPR-associated endonuclease coding sequence including at least one first aptamer sequence and / or a gRNA coding sequence including at least one second aptamer sequence. (Item 73) The lentiviral packaging system according to item 47, wherein the CRISPR-related endonuclease coding sequence comprises at least one aptamer coding sequence. (Item 74) The lentiviral packaging system according to item 47, wherein the gRNA coding sequence comprises at least one aptamer coding sequence. (Item 75) The lentiviral packaging system according to item 74, wherein the at least one aptamer coding sequence is inserted within the tetraloop of the gRNA coding sequence. (Item 76) The lentiviral packaging system described in item 75, wherein the aptamer coding sequence binds to the COM protein. (Item 77) The lentivirus packaging system according to item 72, wherein the at least one first aptamer sequence and the at least one second aptamer sequence are the same aptamer sequence. (Item 78) The lentiviral packaging system according to item 72, wherein the at least one first aptamer sequence is an ABP target RNA binding sequence for a first ABP, the at least one second aptamer sequence is an ABP target RNA binding sequence for a second ABP, and the first and second aptamer sequences are different ABP target RNA binding sequences for first and second ABPs. (Item 79) The lentiviral packaging system according to item 77, wherein the at least one first aptamer sequence and the at least one second aptamer sequence are ABP target RNA binding sequences to ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA binding domain, or COM protein. (Item 80) The lentiviral packaging system according to item 78, wherein the at least one first aptamer sequence and the at least one second aptamer sequence are different ABP target RNA binding sequences for ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N protein RNA binding domain, or COM protein. (Item 81) The lentiviral packaging system according to any one of items 46 to 80, wherein the nonviral nucleic acid sequence includes an RNA stabilizing sequence located at its 3' end. (Item 82) The lentiviral packaging system according to item 81, wherein the RNA stabilization sequence comprises at least one 3'UTR of the human betaglobin gene. (Item 83) The lentiviral packaging system according to any one of items 46 to 82, wherein the plasmid is a lentiviral transfer plasmid. (Item 84) A lentiviral packaging system according to any one of items 46 to 83, wherein at least one of the envelope plasmids comprises an envelope glycoprotein coding sequence encoding an envelope glycoprotein. (Item 85) The lentiviral packaging system according to item 84, wherein the envelope glycoprotein coding sequence encodes VSV-G. (Item 86) A lentiviral packaging system according to any one of items 46 to 85, wherein when the packaging plasmid, the at least one mammalian expression plasmid, and the envelope plasmid are transfected into a eukaryotic cell, the at least one ABP nucleotide sequence does not interfere with viral particle assembly. (Item 87) a) Packaging plasmids that do not encode functional integrase proteins, b) A mammalian expression plasmid comprising at least one eukaryotic cell promoter operably linked to a viral protein R (VPR) coding sequence or a negative regulator (NEF) coding sequence, wherein one or both of the VPR coding sequence or the NEF coding sequence comprises at least one nonviral aptamer-binding protein (ABP) nucleotide sequence, c) A mammalian expression plasmid comprising at least one eukaryotic cell promoter operably ligated to a nonviral nucleic acid sequence, wherein the nonviral nucleic acid sequence comprises a CRISPR-related endonuclease coding sequence, a guide RNA (gRNA) coding sequence, or both a CRISPR-related endonuclease coding sequence and a gRNA coding sequence, d) Envelope plasmid containing an envelope glycoprotein coding sequence and A lentivirus packaging system including [unclear]. (Item 88) A lentiviral packaging system as described in item 87, wherein the nonviral RNA sequence includes at least one aptamer sequence. (Item 89) The lentiviral packaging system according to item 87, wherein the CRISPR-related endonuclease coding sequence comprises at least one aptamer coding sequence. (Item 90) The lentiviral packaging system according to item 87, wherein the gRNA coding sequence comprises at least one aptamer coding sequence. (Item 91) The lenti...

Claims

[Claim 1] The invention as shown in the drawings.

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