WO2019090237A1 - Éditions d'adn sélectivement ciblées chez le bétail - Google Patents

Éditions d'adn sélectivement ciblées chez le bétail Download PDF

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WO2019090237A1
WO2019090237A1 PCT/US2018/059232 US2018059232W WO2019090237A1 WO 2019090237 A1 WO2019090237 A1 WO 2019090237A1 US 2018059232 W US2018059232 W US 2018059232W WO 2019090237 A1 WO2019090237 A1 WO 2019090237A1
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sequence
dna
gene
target
crispr
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PCT/US2018/059232
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English (en)
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Cassandra Joan EDGAR
Andrew Mark CIGAN
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Genus Plc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the field is molecular genetics, and more specifical ly, methods for editing cells of mammals to reducing infection and/or transmission of the viral diseases in mammals,
  • the invention provides methods for using one or more elements/components of a CRISPR-Cas system via a particle delivery formulation and/or system as a means to modify a target polynucleotide.
  • the particle delivery formulation and/or system are nanoparticles.
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide.
  • the CRISPR complex of the invention has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types in various tissues and organs, e.g., endothelial cells, skin, heart, muscle or lung.
  • the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, drag discovery, drag screening, disease diagnosis, and prognosis.
  • the present invention contemplates uses in gene or genome editing, and in reduction in infection and/or transmission of viral diseases in mammals.
  • the modification may occur ex vivo or in vitro, for instance in a cell culture and in some instances not in vivo. In other embodiments, it may occur in vivo.
  • the invention provides a method of modifying an organism or a nonhuman organism by manipulation of a target sequence in a genomic locus of interest comprising: delivering via nanoparticle complex(es) a non-naturally occurring or engineered gene editing bundle composition, wherein the gene editing bundle composition comprises the endonuclease compiexed with at least one guide RNA sequence that is hybridizable to the target sequence, wherein the gene editing bundle composition comprises:
  • An endonuclease and a polynucleotide sequence wherein the polynucleotide sequence comprises at least one guide RNA sequence capable of hybridizing to a target sequence in a eukaryotic cell ; or ⁇ .
  • a polynucleotide sequence encoding an endonuclease wherein the polynucleotide sequence encoding the endonuclease is DNA or RNA, and wherein the polynucleotide sequence further comprises one or more guide sequences, wherein when transcribed, the one or more guide RNA sequences direct sequence-specific binding of the endonuclease to the target sequence.
  • the endonuclease can he constitutively present, or delivered via a nanoparticle, or via a vector expressing the endonuclease, e.g., by sequential or coadministration of a nanoparticle containing or vector containing nucleic acid molecule(s) for in vivo expression of the endonuclease with the nanoparticle containing RNA components of the gene editing system.
  • the nanoparticle can also deliver the vector,
  • the guide RNA sequences can be mutant sequences or the invention can encompass RNA that includes mutant chimeric guide sequences that allow for enhancing performance of these RN As in cells.
  • a suitable promoter such as the Pol III promoter, or such as a U6 promoter, can be added onto the guide RNA that is advantageously delivered via nanoparticle.
  • aspects of the invention also relate to the guide RNA being transcribed in vitro or ordered from a synthesis company and directly transfected. Expression of guide RNAs under the control of the T7 promoter driven by the expression of T7 polymerase in the cell is also envisioned.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a mammal cell .
  • the mammal cell is a porcine cell.
  • the RNA sequence includes the feature.
  • the polynucleotide is DNA and is said to comprise a feature such as a guide RNA sequence
  • the DNA sequence is or can be transcribed into the RNA that comprises the feature at issue.
  • the feature is a protein, such as an endonuclease (for example the CRISPR enzyme)
  • the DNA or RNA sequence referred to is, or can be, translated (and in the case of DNA transcribed first).
  • an RNA encoding an endonuclease is provided to a cell, it is understood that the RNA is capable of being translated by the cell into which it is delivered.
  • the invention provides a method of modifying an organism, e.g., mammal by manipulation of a target sequence in a genomic locus of interest comprising delivering a non-naturally occurring or engineered gene editing bundle composition comprising a nanoparticle, wherein the composition comprises: (A) a non-naturally occurring or engineered gene editing bundle composition comprising a vector system comprising one or more vectors comprising I.
  • a polynucleotide sequence comprising one or more guide sequences.
  • components I and II are delivered together. In other embodiments, components I and II are delivered separately.
  • nanoparticles especially to target heart, muscle or lung tissue or cells, are aspects of the invention.
  • Applicants mean the alteration of the target sequence, which may include the epi genetic manipulation of a target sequence.
  • This epigenetic manipulation may be of the chromatin state of a target sequence, such as by modification of the m ethyl ati on state of the target sequence (i.e. addition or removal of methylation or methylation patterns or CpG islands), hi stone modification, increasing or reducing accessibility to the target sequence, or by promoting 3D folding,
  • the invention provides a method of treating or inhibiting a condition caused by a defect in a target sequence in a genomic locus of interest in a subject (e.g., mammal) in need thereof compri sing modifying the mammal by manipulation of the target sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the target sequence.
  • the invention comprehends delivering an endonuclease comprising delivering to a cell mRNA encoding the CRISPR enzyme, e.g., via nanoparticle complex(es).
  • the CRISPR enzyme is a Cas9.
  • the invention further comprehends nanoparticle complex(es) containing CRISPR complex components or vector(s) for expression, e.g., RNA of the CRISPR complex, or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme), for use in medicine or in therapy, or more generally a method according to the invention, including in vivo, in vitro or ex vivo gene or genome editing.
  • the CRISPR enzyme comprises one or more mutations in one of the catalytic domains.
  • any or all of the polynucleotide sequence encoding the CRISPR enzyme, and the one or more guide sequences, is/are RNA; and advantageously delivered via nanoparticle complex(es).
  • the invention in some embodiments comprehends a method of modifying a genomic locus of interest by minimizing off-target modifications by introducing into a ceil containing and expressing a double stranded DNA molecule encoding a gene product of interest an engineered, non-naturally occurring CRISPR-Cas system, , wherein the introducing is via at least one nanoparticle complex delivering at least a portion of the CRISPR-Cas system (e.g., RNA thereof, and/or the Cas9 and/or a vector encoding the Cas9 and/or a vector expressing the RNA of the system) wherein the system comprises a Cas protein having one or more mutations and at least one guide RNA that targets a DNA molecule respectively, whereby the guide RNA/s target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, where
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with at least one guide sequence hybridized to a target sequence within said target polynucleotide; and advantageously the complex or a component thereof has been delivered via nanoparticle compiex(es).
  • this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises increasing or decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide; and advantageously the complex or a component thereof has been delivered via nanoparticle complex(es).
  • a target polynucleotide can be inactivated to affect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does.
  • a protein or microRNA coding sequence may be inactivated such that the protein or rnicroRNA or pre-microRNA transcript is not produced.
  • Delivery can he in the form of a vector which may be a plasmid or other nucleic acid molecule form, especially when the deliver ⁇ - is via a nanoparticle complex; and the vector also can he viral vector, such as a lenti- or baculo- or preferably adeno-viral/adeno- associated viral vectors, but other means of deliver ⁇ - are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided, especially as to those aspects of the complex not delivered via a nanoparticle complex.
  • viral vector such as a lenti- or baculo- or preferably adeno-viral/adeno- associated viral vectors, but other means of deliver ⁇ - are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided, especially as to those aspects of the complex not delivered via a nanoparticle complex.
  • a vector may mean not only a viral or yeast system (for instance, where the nucleic acids of interest may be operably linked to and under the control of (in terms of expression, such as to ultimately provide a processed RNA) a promoter), but also direct delivery of nucleic acids into a host cell; and advantageously the complex or a component thereof is delivered via nanoparticle complex(es). Also envisaged is a method of delivering the present CRISPR enzyme comprising delivering to a cell mRNA encoding the CRISPR enzyme, and advantageously the complex or a component thereof has been delivered via nanoparticle complex(es).
  • the CRISPR enzyme is truncated, and/or comprised of less than one thousand amino acids or less than four thousand amino acids, and/or is a nuclease or nickase, and/or is codon-optimized, and/or comprises one or more mutations, and/or comprises a chimeric CRISPR enzyme, and/or the other options as herein discussed.
  • AAV and lentiviral vectors are preferred.
  • the target sequence is flanked or followed, at its 3' end, by a PAM suitable for the CRISPR enzyme, typically a Cas and in particular a Cas9.
  • a PAM suitable for the CRISPR enzyme typically a Cas and in particular a Cas9.
  • a suitable PAM is 5'-NGG or 5 -N GRR for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively.
  • the invention provides nanoparticle formulation comprising one or more guide RNAs are delivered in vitro, ex vivo or in vivo in the context of the CRISPRCas system.
  • the guide RNA- nanoparticle formulations may be delivered separately from the Cas9.
  • a dual-deliver ⁇ ' system is envisaged such that the Cas 9 may be delivered via a vector and the guide RN As are provided in a nanoparticle formulation, where vectors are considered in the broadest light as simply any means of delivery, rather than specifically viral vectors.
  • separate delivery of the guide RNA-nanop article formulation and the Cas9 may be sequential, for example, first Cas9 vector is delivered via a vector system followed by delivery of sgR A- nanoparticle formulation) or the sgRNA-nanoparticle formulation and Cas9 may be delivered substantially contemporaneously (i.e., co-delivery). Sequential delivery may he done at separate points in time, separated by days, weeks or even months.
  • multiple guide RNAs formulated in one or more deliver ⁇ ' vehicles may be provided with a Cas9 delivery system.
  • the Cas9 is also delivered in a nanoparticle
  • the guide RNA-nanoparticle formulation and the Cas9 nanoparticle formulation may be delivered separately or may be delivered substantially contemporaneously (i.e., co-delivery). Sequential delivery could be done at separate points in time, separated by days, weeks or even months.
  • nanoparticle formulations comprising one or more guide RNAs are adapted for delivery in vitro, ex vivo or in vivo in the context of the CRISPR- Cas system to different target genes, different target cells or different target different tissues/organs, e.g., heart or muscle or lung.
  • Multiplexed gene targeting using nanoparticle formulations comprising one or more guide RNAs are also envisioned,
  • a nanoparticle formulation comprising one or more components of the CRISPR-Cas system is provided.
  • a gRNA-nanoparticle formulation comprising one or more guide RNAs is provided.
  • a method of treating a subject suffering from a disease or disorder associated with any tissue or organ comprising administering a composition comprising a nanoparticle formulation comprising one or more components of the CRISPR- Cas system.
  • sgRNA may be pre-complexed with the Cas9 protein, before formulating the entire complex in a particle.
  • Formulations may he made with a different molar ratio of different components known to promote delivery of nucleic acids into ceils (e.g.
  • DOTAP 1,2-ditetradecanoyl-sn- glycero-3- phosphocholine
  • PEG polyethylene glycol
  • cholesterol 1,2-ditetradecanoyl-sn- glycero-3- phosphocholine
  • DMPC 1,2-ditetradecanoyl-sn- glycero-3- phosphocholine
  • PEG polyethylene glycol
  • cholesterol cholesterol
  • DMPC 1,2-ditetradecanoyl-sn- glycero-3- phosphocholine
  • PEG polyethylene glycol
  • cholesterol cholesterol
  • the invention accordingly comprehends admix
  • the invention in an embodiment comprehends a method of preparing an sgRNA-and Cas9 protein containing particle comprising admixing an sgRNA and Cas9 protein mixture with a mixture comprising or consisting essentially of or consisting of surfactant, phospholipid, biodegradable polymer, lipoprotein and alcohol.
  • An embodiment comprehends an sgRNA-and Cas9 protein containing particle from the method.
  • the invention in an embodiment comprehends use of the particle in a method of modifying a genomic locus of interest, or an mammal by mampulation of a target sequence in a genomic locus of interest, comprising contacting a cell containing the genomic locus of interest with the particle wherein the sgRNA targets the genomic locus of interest; or a method of modifying a genomic locus of interest, or mammal by manipulation of a target sequence in a genomic locus of interest, comprising contacting a cell containing the genomic locus of interest with the particle wherein the sgRNA targets the genomic locus of interest.
  • the invention is used to prevent infection and/or transmission of viral diseases in mammals.
  • the invention is used to prevent infection and/or transmission of the PRRS virus.
  • Viral infections are a major source of morbidity and mortality in the livestock industry.
  • porcine reproductive and respiratory syndrome virus PRRSV
  • PRRSV porcine reproductive and respiratory syndrome virus
  • the disease can be found in virtually every area of the world where swine are raised, namely Europe, North America, China, Japan, Vietnam, the Philippines, Malaysia, Korea, and others.
  • PRRSV has become one of the most significant diseases in intensively raised pigs. About 45% of these losses stem from reproductive failure in adult females, and the remaining losses occur as a result of respiratory disease, especially pneumonia in nursing pigs.
  • Porcine reproductive and respiratory syndrome virus is a virus that causes a disease of pigs, called porcine reproductive and respiratory syndrome(PRRS), also known as blue-ear pig disease (in Chinese, zhii Idner hing 3 ⁇ 4 ' 1 ⁇ 5 ⁇ 3 ⁇ 4).
  • PRRS porcine reproductive and respiratory syndrome
  • PRRS is caused by a vims which was first isolated and classified as an arterivirus as recently as 1991.
  • the disease syndrome had been first recognized in the USA in the mid 1980's and was called "mystery swine disease”. It has also been called blue ear disease.
  • the name porcine arterivirus has been proposed recently.
  • PRRSV Porcine Reproductive and Respiratory Syndrome Virus
  • PRRSv Porcine Reproductive and Respiratory Syndrome Virus
  • PRRSv is a member of the mammalian arterivirus group, which share pathogenic traits, including macrophage tropism and the ability to cause serious disease and lasting infection.
  • PRRSv results in respiratory illness, including cough and fever and lowered growth performance.
  • PRRSv infection often results in reproductive failure, as well as chronically infected and low birth weight piglets.
  • Vaccines have not been effective against the disease, and although genetic selection for natural resistance is an option, success to date has been limited, possibly due to the genetic diversity of the virus.
  • the PRRS virus has therefore become established in most swine-producing regions of the world, with only a few exceptions. It is generally assumed that 60-80% of production herds are typically infected.
  • the virus of PRRS has a particular affinity for the macrophages particularly those found in the lung. Macrophages are part of the body defenses. Those present in the lung are called alveolar macrophages. They ingest and remove invading bacteria and viruses but not in the case of the PRRS virus. Instead, the virus multiplies inside them producing more virus and kills the macrophages. Once it has entered a herd it tends to remain present and active indefinitely. Up to 40% of the macrophages are destroyed which removes a major part of the bodies defense mechanism and allows bacteria and other viruses to proliferate and do damage.
  • a common example of this is the noticeable increase in severity of enzootic pneumonia in grower/finisher units when they become infected with PRRS virus.
  • sow herds 1 -2 years after infection may contain less than 20% of serological positive animals. This does not however necessarily mean they are not still immune, nor does it mean that they have stopped passing on immunity to their offspring.
  • Adult animals shed virus for much shorter periods of time (14 days) compared to growing pigs which can excrete for 1-2 months.
  • the clinical picture can vary tremendously from one herd to another. As a guide, for every three herds that are exposed to PRRS for the first time one will show no recognizable disease, the second would show mild disease and the third moderate to severe disease. The reasons for this are not clearly understood. However, the higher the health status of the herd, the less severe are the disease effects. It may be that the virus is mutating as it multiplies, throwing up some strains that are highly virulent and some that are not.
  • PRRS infects all types of herd including high or ordinary health status and both indoor and outdoor units, irrespective of size.
  • Clinical signs in farrowing sows in the first month of infection include inappetence over the farrowing period, a reluctance to drink, absence of milk (agalactia), mastitis, discoloration of the skin and pressure sores associated with small vesicles, lethargy, respirator ⁇ - signs, mummified piglets (10-15% may die in the last 3-4 weeks of pregnancy), stillbirth levels (increase up to 30%), and very weak piglets at birth.
  • the initial phase of inappetence and fever will often take 3-6 weeks to move through. Coughing occurs in some sows and a few individual cases of clinical pneumonia may occur.
  • This acute phase lasts in the herd for up to 6 weeks, and is characterized by early farrowings, increases in stillbirths, weak pigs and an increase in the numbers of large mummified pigs that have died in the last three weeks of pregnancy. In some herds, these may reach up to 30% of the total pigs born. Piglet mortality peaks at 70% in weeks 3 or 4 after the onset of symptoms and only returns to pre-infected levels after 8-12 weeks. The reproductive problems may persist for 4-8 months before returning to normal, however in some herds it may actually improve on the pre-PRRS performance.
  • Clinical signs in piglets include less viable piglets and increase in respirator ⁇ - infections such as giassers disease.
  • Clinical signs in boars include inappetence, increased body temperature, lethargy, loss of libido, lowered fertility, poor litter sizes, and lowered sperm output.
  • Diagnosis is based on the clinical signs, post mortem examinations and the known presence of the virus in the herd or by serological examinations and isolation of the virus in a laboratory.
  • Genome engineering includes altering the genome by deleting, inserting, mutating, or substituting specific nucleic acid sequences.
  • the alteration can be gene- or location-specific.
  • Genome engineering can use site-directed nucleases, such as Cas proteins and their cognate polynucleotides, to cut DNA, thereby generating a site for alteration.
  • the cleavage can introduce a double-strand break (DSB) in the DNA target sequence.
  • DSBs can be repaired, e.g., by non-homologous end joining (NHEJ),
  • HDR homology-directed repair
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated proteins
  • the CRISPR-Cas system provides adaptive immunity against foreign DNA in bacteria (see, e.g., Barrangou, R., et al, Science 315: 1709-1712 (2007); Makarova, . S., et al., Nature Reviews Microbiology 9:467-477 (2011); Garneau, J. E., et al., Nature 468:67-71 (2010); Sapranauskas, R., et al., Nucleic Acids Research 39:9275-9282 (201 1)).
  • CRISPR-Cas systems have recently been reclassified into two classes, comprising five types and sixteen subtypes (see Makarova, K., et al,, Nature Reviews Microbiology 13 : 1-15 (2015)). This classification is based upon identifying all Cas genes in a CRISPR-Cas locus and determining the signature genes in each CRISPR-Cas locus, ultimately placing the CRISPR-Cas systems in either Class 1 or Class 2 based upon the genes encoding the effector module, i.e., the proteins involved in the interference stage.
  • Class 1 systems have a multi-subunit crRNA-effector complex
  • Class 2 systems have a single protein, such as Cas9, Cpfl, C2cl, C2c2, C2c3, or a crRNA-effector complex
  • Class 1 systems comprise Type I, Type III, and Type IV systems
  • Class 2 systems comprise Type II, Type V, and Type VI systems.
  • Type II systems have casl, cas2, and cas9 genes.
  • the cas9 gene encodes a multi-domain protein that combines the functions of the crRNA-effector complex with DNA target sequence cleavage.
  • Type II systems are further divided into three subtypes, subtypes II- A, II-B, and II-C.
  • Subtype II- A contains an additional gene, csn2. Examples of organisms with a subtype It-A systems include, but are not limited to, Streptococcus pyogenes,
  • Subtype II-B lacks the csn2 protein but has the cas4 protein.
  • An example of an organism with a subtype II-B system is Legionella pneumophila.
  • Subtype II-C is the most common Type II system found in bacteria and has only three proteins, Casl, Cas2, and Cas9.
  • An example of an organism with a subtype II-C system is Neisseria lactamica.
  • Type V systems have a cpfl gene and casl and cas2 genes (see Zetsche, B., et al., Cell 163 : 1-13 (2015)).
  • the cpfl gene encodes a protein, Cpfl, that has a RuvC-like nuclease domain that is homologous to the respective domain of Cas9 but lacks the HNH nuclease domain that is present in Cas9 proteins.
  • Type V systems have been identified in several bacteria including, but not limited to, Parcubacteria bacterium, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Acidaminococcus spp., Porphyromonas macacae, Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi, Smithella spp., Leptospira inadai, Franciscelia tuiarensis, Franciscella novicida, Candidatus methanoplasma termitum, and Eubacterium eligens.
  • Cpfl also has RNase activity and is responsible for pre-crRNA processing (see Fonfara, I, et al., Nature 532(7600):517-521 (2016)).
  • the crRNA is associated with a single protein and achieves interference by combining nuclease activity with RNA-binding domains and base-pair formation between the crRNA and a nucleic acid target sequence.
  • nucleic acid target sequence binding involves Cas9 and the crRNA, as does nucleic acid target sequence cleavage.
  • the RuvC-like nuclease (RNase H fold) domain and the HNH (McrA-like) nuclease domain of Cas9 each cleave one of the strands of the double-stranded nucleic acid target sequence.
  • the Cas9 cleavage activity of Type II systems also requires hybridization of crRNA to a tracrRNA to form a duplex that facilitates the crRNA and nucleic acid target sequence binding by the Cas9 protein.
  • RNA-guided Cas9 endonuclease has been widely used for programmable genome editing in a variety of organisms and model systems (see, e.g., Jinek M., et ai., Science 337:816-821 (2012), Jinek M,, et al., eLife 2:e00471 , doi: 10.7554/eLife.00471 (201.3); U.S. Published Patent Application No. 2014-0068797, published 6 Mar. 2014).
  • nucleic acid target sequence binding involves Cpfl and the crRNA, as does nucleic acid target sequence cleavage.
  • the RuvC-like nuclease domain of Cpfl cleaves one strand of the double-stranded nucleic acid target sequence
  • a putative nuclease domain cleaves the other strand of the double-stranded nucleic acid target sequence in a staggered configuration, producing 5 ! overhangs, which is in contrast to the blunt ends generated by Cas9 cleavage.
  • the Cpfl cleavage activity of Type V systems does not require hybridization of crRNA to tracrRNA to form a duplex, rather the crRNA of Type V systems uses a single crRNA that has a stem-loop structure forming an internal duplex.
  • Cpfl binds the crRNA in a sequence and structure specific manner that recognizes the stem loop and sequences adjacent to the stem loop, most notably the nucleotides 5 ! of the spacer sequences that hybridizes to the nucleic acid target sequence.
  • This stem-loop structure is typically in the range of 15 to 19 nucleotides in length.
  • nucleotides 5' of the stem loop adopt a pseudo-knot structure further stabilizing the stem-loop structure with non-canonical Watson-Crick base pairing, triplex interaction, and reverse Hoogsteen base pairing (see Yam an o, T., et al., Cell 165(4):949-962 (2016)).
  • the crRNA forms a stem-loop structure at the 5' end, and the sequence at the 3' end is complementary to a sequence in a nucleic acid target sequence.
  • C2cl Class 2 candidate 1
  • C2c3 Class 2 candidate 3
  • C2cl and C2c3 proteins are similar in length to Cas9 and Cpfl proteins, ranging from approximately 1,100 amino acids to approximately 1,500 amino acids.
  • C2cl and C2c3 proteins also contain RuvC-Iike nuclease domains and have an architecture similar to Cpfl .
  • C2c proteins are similar to Cas9 proteins in requiring a crRNA and a tracrRNA for nucleic acid target sequence binding and cleavage but have an optimal cleavage temperature of 50. degree. C.
  • C2cl proteins target an AT-rich protospacer adjacent motif (PAM), similar to the PAM of Cpfl, which is 5 ! of the nucleic acid target sequence (see, e.g., Shmakov, S., et al, Molecular Cell 60(3):385-397 (2015)),
  • PAM AT-rich protospacer adjacent motif
  • Class 2 candidate 2 (C2c2) does not share sequence similarity with other CRISPR effector proteins and was recently identified as a Type VI system (see Abudayyeh, O., et al., Science 353(6299):aaf5573 (2016)).
  • C2c2 proteins have two HEPN domains and demonstrate single-stranded RNA cleavage activity, C2c2 proteins are similar to Cpfl proteins in requiring a crRNA for nucleic acid target sequence binding and cleavage, although not requiring tracrRNA. Also, similar to Cpfl, the crRNA for C2c2 proteins forms a stable hairpin, or stem-loop structure, that aids in association with the C2c2 protein.
  • Type VI systems have a single polypeptide RNA endonuclease that utilizes a single crRNA to direct site-specific cleavage. Additionally, after hybridizing to the target RNA complementary to the spacer, C2c2 becomes a promiscuous RNA endonuclease exhibiting non-specific endonuclease activity toward any single-stranded RNA in a sequence independent manner (see East-Seletsky, A., et al., Nature 538(7624):270-273 (2016)).
  • Cas9 orthologs are known in the art as well as their associated polynucleotide components (tracrRNA and crRNA) (see, e.g., Fonfara, I, et al., Nucleic Acids Research 42(4):2577-2590 (2014), including all Supplemental Data; Chyiinski K., et al., Nucleic Acids Research 42(10):6091-6105 (2014), including all Supplemental Data).
  • Cas9 ⁇ !ike synthetic proteins are known in the art (see U.S. Published Patent Application No. 2014-0315985, published 23 Oct. 2014).
  • Cas9 is an exemplary Type II CRISPR Cas protein.
  • Cas9 is an endonuclease that can be programmed by the tracrRNA/crRNA to cleave, in a site-specific manner, a DNA target sequence using two distinct endonuclease domains (HNH and RuvC/RNase H-like domains) (see U.S. Published Patent Application No. 2014-0068797, published 6 Mar. 2014; see also Jinek, M , et al., Science 337:816-821 (2012)),
  • each wild-type CRISPR-Cas9 system includes a crRNA and a tracrRNA,
  • the crRNA has a region of complementarity to a potential DNA target sequence and a second region that forms base-pair hydrogen bonds with the tracrRNA to form a secondary structure, typically to form at least one stem structure.
  • tracrRNA and a crRNA interact through a number of base-pair hydrogen bonds to form secondary RNA structures.
  • Complex formation between tracrRNA/crRNA and Cas9 protein results in conformational change of the Cas9 protein that facilitates binding to DNA, endonuclease activities of the Cas9 protein, and crRNA-guided site-specific DNA cleavage by the endonuclease Cas9.
  • the DNA target sequence is adjacent to a cognate PAM.
  • the complex can be targeted to cleave at a locus of interest, e.g., a locus at which sequence modification is desired.
  • the spacer of Class 2 CRISPR-Cas systems can hybridize to a nucleic acid target sequence that is located 5' or 3' of a PAM, depending upon the Cas protein to be used.
  • a PAM can vary depending upon the Cas polypeptide to be used.
  • the PAM can be a sequence in the nucleic acid target sequence that comprises the sequence 5'-NRR-3', wherein R can be either A or G, N is any nucleotide, and N is immediately 3' of the nucleic acid target sequence targeted by the nucleic acid target binding sequence.
  • a Cas protein may be modified such that a PAM may be different compared with a PAM for an unmodified Cas protein. If, for example, Cas9 from S.
  • the Cas9 protein may be modified such that the PAM no longer comprises the sequence 5'-NGG-3', but instead comprises the sequence 5'-NNR-3', wherein R can be either A or G, N is any nucleotide, and N is immediately 3 ! of the nucleic acid target sequence targeted by the nucleic acid target sequence.
  • Cpfl has a thymine-rich PAM site that targets, for example, a TTTN sequence (see Fagerlund, R, et al., Genome Biology 16:251 (2015)),
  • range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from I to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 11 ⁇ 2, and 43 ⁇ 4 This applies regardless of the breadth of the range.
  • gene-editing refers to the use of naturally occurring or artificially engineered nucleases, also referred to as “molecular scissors.”
  • the nucleases create specific double- stranded break (DSBs) at desired locations in the genome, which in some cases harnesses the cell's endogenous mechanisms to repair the induced break by natural processes of
  • a protein containing a nuclease domain can bind and cleave a target nucleic acid by forming a complex with a nucleic acid-targeting nucleic acid.
  • the cleavage can introduce double stranded breaks in the target nucleic acid.
  • a nucleic acid can be repaired e.g. by endogenous non-homologous end joining (NHEJ) machinery.
  • NHEJ non-homologous end joining
  • a piece of nucleic acid can be inserted.
  • nucl eic acid-targeting nucl eic aci ds and site-directed polypeptides can introduce new functions to be used for genome editing.
  • a nuclease used for gene editing is CRISPR-Cas9.
  • Other examples are meganucleases, zinc finger nucleases, TALENS, and other CRISPR variants.
  • homoing DNA technology covers any mechanisms that allow a specified molecule to be targeted to a specified DNA sequence including Zinc Finger (ZF) proteins, Transcription Activator-Like Effectors (TALEs) meganucleases, and the CRISPR/Cas9 system.
  • ZF Zinc Finger
  • TALEs Transcription Activator-Like Effectors
  • genetically altered means those animals or embryos or cells which have a desired genetic modification such as a knock-out, knock-in, conditional, inducible, transient or point mutation(s) of any gene or its regulatory mechanism or a transgenic with foreign or modified gene/s or regulatory sequences, or having undergone genomic modification in any way including but not limited to recombination, chromosomal deletion, addition, translocation, rearrangement or addition, deletion or modification of nucleic acid, protein or any other natural or synthetic molecule or organelle, or cytoplasmic or nuclear transfer, leading to inheritable changes.
  • Knock-in means replacement of an endogenous gene with a transgene or with same endogenous gene with some structural modification/s but retaining the transcriptional control of the endogenous gene.
  • Knock-out means disruption of the structure or regulatory mechanism of a gene. Knock-outs may be generated through homologous recombination of targeting vectors, replacement vectors or hit-and-run vectors or random insertion of a gene trap vector resulting into complete, partial or conditional loss of gene function.
  • a "binding protein” is a protein that is able to bind to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an R ' NA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a DNA-binding protein a DNA-binding protein
  • RNA molecule an R ' NA-binding protein
  • a protein-binding protein a protein-binding protein.
  • a binding protein can have more than one type of binding activity.
  • zinc finger proteins have DNA-binding, RNA-binding and protein- binding activity.
  • a "zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • a "TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence, A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein.
  • Zinc finger and TALE binding domains can be "engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of naturally occurring zinc finger or TALE proteins.
  • engineered DNA binding proteins are proteins that are non-naturally occurring.
  • methods for engineering DNA- binding proteins are design and selection.
  • a designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos. 6, 140,081; 6,453,242, and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No. 20110301073,
  • a "selected" zinc finger protein or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. No. 5,789,538; U.S. Pat. No.
  • Cleavage refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double- stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events, DN A cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion
  • polypeptides are used for targeted double-stranded DNA cleavage.
  • a "cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).
  • first and second cleavage half- domains;" “+ and - cleavage half-domains” and “right and left cleavage half-domains” are used interchangeably to refer to pairs of cleavage half-domains that dimerize.
  • An "edited cleavage half-domain” is a cleavage half-domain that has been modified so as to form obligate heterodimers with another cleavage half-domain (e.g., another engineered cleavage half-domain).
  • Means for generating a double strand DNA break refers to a molecular structure that is capable of cleaving both strands of a double-stranded DNA molecule.
  • Such structures include polypeptide domains comprised within many known nuclease proteins, for example, the Fokl nuclease domain, the catalytic domain is selected from the group consisting of proteins Mmel, Colicin-E7 (CEA7JECOLX), Colicin-E9, APFL, EndA, Endo I (ENDIJEC0LI), Human Endo G ( N L ' C ' G H UM AN ).
  • Bovine Endo G (NUCG _ BOVIN), R.HinPl 1, 1-Bas-I, I- Brno-I, 1-Hmul, 1-Tev-l , 1 -Tevll, 1-Tevll 1, 1 - Twol, R.Mspl, R.Mval, NucA, NucM, Vvn, Vvn CLS, Staphylococcal nuclease (NUC STAAU), Staphylococcal nuclease
  • Endodeoxyribonuclease I Endodeoxyribonuclease I (ENRNJBPTT), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD61 (R.BspD61 large subunit), ss.BspD61 (R.BspD61 small sub unit), R.Plel, Mlyl, Alwl, Mval2691, Bsrl, Bsml, Nb.BtsCL Nt.BtsCI, Rl.Btsl, R2.Btsl, BbvCI subunit 1 , BbvCI subunit 2, BpulOI alpha subunit, BpulOI beta subunit, Bmrl, Bfil, 1 -Crel, hExol
  • EXOIJHUMAN Yeast Exol
  • EX0I_YEAST E.coli Exol
  • Human TREX2 Mouse TREXI, Human TREXI, Bovine TREXI, Rat TREXI, Human DNA2, Yeast DNA2 (DNA2 YEAST).
  • Means for repairing a double strand DNA break refers to a molecular structure that is capable of facilitating/catalyzing the joining of the ends of double-stranded DNA molecules, for example, by joining ends generated by cleaving a single double-stranded DNA molecule, or by joining one end generated by cleaving a single double-stranded DNA molecule with the end of an exogenous double-stranded DNA molecule.
  • Such structures include polypeptide domains comprised within many known ligase proteins, for example, Cre recombinase.
  • the same molecular structure may serve as both a means for generating a double strand DNA break and a means for repairing a double strand DNA break, where the same structure facilitates both the cleavage and repair of double-stranded DNA molecules (e.g., Hin recombinase).
  • the induction of the site specific double stranded breaks in the genome induces the host cell DNA repair pathway which resolves the double stranded break through homology-directed repair (HDR) or non-homologous end-joining (NHEJ) repair.
  • HDR homology-directed repair
  • NHEJ non-homologous end-joining
  • the donor polynucleotide can be DNA or RNA, single-stranded and/or double-stranded and can be introduced into a ceil in linear or circular form.
  • embodiments of the present invention may also include linear exogenous (donor) nucleic acid(s), compositions comprising these nucleic acids and methods of making and using these linear donor molecules.
  • the linear donor molecule stably persists in the cell into which it is introduced.
  • the linear donor molecule is modified to resist exonucieolytic cleavage, for example by placing one or more phosphorothioate phosphodiester bonds between one or more base pairs on the ends of the donor molecule.
  • the linear exogenous nucleic acid may also include single stranded specific DNA.
  • CRISP associated protein refers to a protein family that is strictly associated with CRISPR elements and always occur near a repeat cluster of in CRISPR segments.
  • Cas proteins include but are not limited to Cas9 family member proteins, Cas6 family member proteins (e.g., Csy4 and Cas6), and Cas5 family member proteins. Examples of Cas protein families and methods of identifying the same have been disclosed in Haft, Daniel H., et al., 2005. A Guild of 45 CRISPR- Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes. PLoS Compiit. Bio. 1(6): e60, which is incorporated by reference herein in its entirety.
  • Cas9 can generally refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild-type Cas9 polypeptide (e.g., Cas9 from S. pyogenes).
  • Illustrative Cas9 sequences are provided by SEQ ID Nos. 1-256 and 795-1346 of U.S.
  • Cas9 can refer to can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild type Cas9 polypeptide (e.g., from S. pyogenes).
  • Cas9 can refer to the wild-type or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
  • Cas5 can generally refer to can refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild type illustrative Cas5 polypeptide (e.g., Cas5 from D. vulgaris).
  • Illustrative Cas5 sequences are provided in Figure 42 of U.S. Patent Publication No, 2016/0046963.
  • Figure 42 of U.S. Patent Publication No. 2016/0046963 is hereby incorporated herein by reference.
  • Cas5 can generally refer to can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or !0Q% sequence identity and/or sequence similarity to a wild-type Cas5 polypeptide (e.g., a Cas5 from D. vulgaris).
  • Cas5 can refer to the wild-type or a modified form of the Cas5 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
  • Cas6 can generally refer to can refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild type illustrative Cas6 polypeptide (e.g., a Cas6 from T. thermophihs).
  • Illustrative Cas6 sequences are provided in Figure 41 of U.S. Patent Publication No. 2016/0046963.
  • Figure 41 of U.S. Patent Publication No. 2016/0046963 is hereby incorporated herein by reference.
  • Cas6 can generally refer to can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild-type Cas6 polypeptide (e.g., from T. thermophilics).
  • Cas6 can refer to the wiicltype or a modified form of the Cas6 protein that ca comprise a amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
  • CRISPR/Cas9 or “CRISPR/Cas9 system” refer to a
  • programmable nuclease system for genetic editing that includes a Cas9 protein, or derivative thereof, and one or more non-coding RNAs that can provide the function of a CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA) for the Cas9.
  • the crRNA and tracrRNA can be used individually or can be combined to produce a "guide RNA" (gRNA).
  • the crRNA or gRNA provide sequence that is complementary to the genomic target.
  • Cpfl refers to another programmable RNA-guided endonuclease of a class 2 CRISPR-Cas system, described and used for gene editing purposes (Zetsche et al., Cell 163 :759-771, 2015). This system uses a non-specific endonuclease unit from the Cpfl protein family, with a specificity of cleavage conferred by a single crRNA (lacking tracer RNA). Similar to Cas9, the Cpfl coding sequence can be fused to UTR sequences described herein to improve its stability, and thus the efficiency of the resulting gene editing method,
  • Resistance or "disease resistance” refers to the extent to which an organism can defend itself from and/or withstand the attack of a pathogen and remain unaffected.
  • An organism may demonstrate complete resistance, meaning it remains virtually unaffected by a pathogen.
  • an organism may demonstrate partial resistance, wherein the extent to which the pathogen affects the organisms is less than a comparable organism with no resistance. Resistance may stem from a particular characteristic of the organism, allowing the organism to avoid the outcome of organism-pathogen interactions. Resistance is
  • increased resistance and reduced susceptibility refer to a statistically significant reduction of the incidence and/or severity of clinical signs or clinical symptoms which are associated with infection by a given pathogen.
  • increased resistance or reduced susceptibility can refer to a statistically significant reduction of the incidence and/or severity of clinical signs or clinical symptoms which are associated with infection by PRRSV in an animal comprising a deleted or inactivated chromosomal sequence in a CD 163 gene protein as compared to a control animal having an unmodified chromosomal sequence.
  • statically significant reduction of clinical symptoms means, but is not limited to, the frequency in the incidence of at least one clinical symptom in the modified group of subjects is at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50%, and even more preferably at least 70% lower than in the non- modified control group after the challenge with the infectious agent.
  • the terms "reduction of the incidence and/or severity of clinical signs” or “reduction of clinical symptoms” mean reducing the number of infected subjects in a group, reducing or eliminating the number of subjects exhibiting clinical signs of infection, or reducing the severity of any clinical signs that are present in one or more subjects, in comparison to wild-type infection.
  • these terms encompass any clinical signs of infection, lung pathology, viremia, antibody production, reduction of pathogen load, pathogen shedding, reduction in pathogen transmission, or reduction of any clinical sign symptomatic of PRRSV.
  • these clinical signs are reduced in one or more animals of the invention by at least 10% in comparison to subjects not having a modification in the CDl 63 gene and that become infected. More preferably clinical signs are reduced in subjects of the invention by at least 20%, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%.
  • breeding refers to the union of male and female gametes so that fertilization occurs. Such a union may be brought about by mating
  • Such artificial methods include, but are not limited to, artificial insemination, surgical assisted artificial insemination, in vitro fertilization, intracytoplasmic sperm injection, zona drilling, in vitro culture of fertili zed oocytes, ovary transfer, and ovary splitting.
  • breeding as used herein also includes transferring of a fertilized oocyte into the reproductive tract of a female animal.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Optimal alignment of sequences for comparison can use any means to analyze sequence identity (homology) known in the art, e.g., by the progressive alignment method of termed "PILEUP" (Morrison, Mol. Biol. Evol. 14:428-441 (1997), as an example of the use of PILEUP); by the local homology algorithm of Smith & Waterman (Adv. Appl. Math. 2: 482 ( 1981 )); by the homology alignment algorithm of Needleman & Wunsch (J. Mol. Biol. 48:443 (1970)); by the search for similarity method of Pearson (Proc. Natl. Acad. Sci. USA 85: 2444 (1988)), by computerized implementations of these algorithms (e.g., GAP, BEST FIT, FASTA, and TFASTA in the Wisconsin Genetics
  • BLAST algorithm Another example of algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschui et al, J. Mol. Biol. 2 5: 403-410 (1990).
  • the BL AST programs (Basic Local Alignment Search Tool) of Altschui, S. F., et al., (1993) J. Mol. Biol. 215:403-410) searches under default parameters for identity to sequences contained in the BLAST "GENEMBL" database.
  • a sequence can be analyzed for identity to all publicly available DNA sequences contained in the GENEMBL database using the BLASTN algorithm under the default parameters.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschui et al, J. Mol. Biol. 215: 403-410 (1990)).
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below, due to the
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • W word length
  • B BLOSUM62 scoring matrix
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1 , more preferably less than about 0.01, and most preferably less than about 0.001.
  • GAP Global Alignment Program
  • GAP uses the algorithm of Needleman and Wunsch J. Mol. Biol.
  • a general purpose scoring system is the BLOSUM62 matrix (He ikoff and Henikoff, Proteins, 17: 49-61 ( 1993)), which is currently the default choice for BLAST programs.
  • BLOSUM62 uses a combination of three matri ces to cover all contingencies. Altschul, J. Mol. Biol. 36: 290-300 (1993), herein incorporated by reference in its entirety and is the scoring matrix used in Version 10 of the Wisconsin Package®
  • sequence identity or “identity” in the context of two nucleic acid sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • Identity to a sequence used herein would mean a polynucleotide sequence having at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80%> identity, more preferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • CRISPR Since its initial use as a gene editing tool less than a decade ago, CRISPR has become a very attractive prospect to researchers looking to investigate or alter the structure of an organism's genome.
  • the technology's potential in treating a host of genetic diseases mean that it has been of particular interest within the medical community, but as many people have found out, CRISPR in practice is not always that easy and are many problems that have to be overcome before CRISPR can be considered as a therapeutic option.
  • One of these problems is our limited ability to introduce the components of the CRISPR ⁇ Cas9 system to the nucleus of the cell so that it can perform the desired edits.
  • the system is made up of two different molecular components: a protein called Cas9 which can cleave DNA, and an RNA molecule called gRNA (or sgRNA) that guides the protein to the right locus in the genome. If either of these components are not present in the nucleus, gene editing cannot be achieved, and the reaction will fail. In the past, attempts at introducing them both have triggered the natural defenses of the cell, 'trapping' the components before they can reach the nucleus and significantly reducing the treatment potential.
  • Genome editing through the deliver ⁇ ' of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies.
  • Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge.
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic ceil.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e,g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • a "disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown and may he at a normal or abnormal level.
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • a gene product e.g., a protein
  • a non-coding sequence e.g., a regulatory polynucleotide or a junk DNA.
  • PAM protospacer adjacent motif
  • the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence).
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • Similar considerations and conditions apply as above for methods of modifying a target polynucleotide. In fact, these sampling, culturing and re-introduction options apply across the aspects of the present invention.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or nonhuman animal and modifying the cell or cells.
  • This invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to the CRISPR-Cas system and components thereof as delivered by nanoparticle carrier.
  • sequence targeting such as genome perturbation or gene-editing
  • An advantage of the present methods is that the delivery of the CRISPR system can be directed to minimize off-target deliver ⁇ ' and its resulting side effects. This is achieved using deliver ⁇ ' particle formulations and/or systems, preferably, nanoparticle systems.
  • CRISPR complex e.g., CRISPR enzyme or mRNA or guide RNA delivered using
  • nanoparticles or lipid envelopes Discussion herein as to other delivery systems or vectors are because such may be used in conjunction with the nanoparticle aspects of the invention.
  • nanoparticle refers to any particle having a diameter of less than 1000 nm.
  • nanoparticles of the invention have a greatest dimension (e.g., diameter) of 500 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 25 nm and 200 nm.
  • nanoparticles of the invention have a greatest dimension of 100 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 35 nm and 60 nm.
  • Nanoparticles encompassed in the present invention may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof
  • Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles).
  • Nanoparticles made of semiconducting material may also he labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present invention.
  • a particle delivery system/formulation is defined as any biological deliver ⁇ ' system/formulation which includes a nanoparticle in accordance with the present invention.
  • a particle in accordance with the present invention is any entity having a greatest dimension (e.g. diameter) of less than 100 microns ( ⁇ ).
  • inventive particles have a greatest dimension of less than 10 microns ( ⁇ ).
  • inventive particles have a greatest dimension of less than 2000 nanometers (nm).
  • inventive particles have a greatest dimension of less than 1000 nanometers (nm).
  • inventive particles have a greatest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.
  • inventive particles have a greatest dimension (e.g., diameter) of 500 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 250 nm or less. In some embodiments, inventive particles have a greatest dimension ( e.g., diameter) of 200 nm or less. In some embodiments, inventive particles have a greatest dimension ( e.g., diameter) of 150 nm or less. In some embodiments, inventive particles have a greatest dimension ( e.g., diameter) of 100 nm or less. Smaller nanoparticles, e.g., having a greatest dimension of 50 nm or less are used in some embodiments of the invention. In some embodiments, inventive particles have a greatest dimension ranging between 25 nm and 200 nm.
  • Particle characterization is done using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transfoml infrared spectroscopy (FTIR), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarisation interferometry and nuclear magnetic resonance (NMR).
  • TEM electron microscopy
  • AFM atomic force microscopy
  • DLS dynamic light scattering
  • XPS X-ray photoelectron spectroscopy
  • XRD powder X-ray diffraction
  • FTIR Fourier transfoml infrared spectroscopy
  • MALDI-TOF matrix-assisted laser desorption/ionization time of flight mass spectrometry
  • Characterization may be made as to native particles (i.e., preloading) or after loading of the cargo (herein cargo refers to e.g., one or more components of CRISPR-Cas system e.g., CRISPR enzyme or mRNA or guide RNA, or any combination thereof and may include additional carriers and/or excipients) to provide particles of an optimal size for delivery for any in vitro, ex vivo and/or in vivo application of the present invention.
  • particle dimension (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS).
  • DLS dynamic laser scattering
  • Particles delivery systems within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles.
  • any of the delivery systems described herein including but not limited to, e.g., lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene gun may be provided as particle delivery systems within the scope of the present invention,
  • CRISPR complex e.g., CRISPR enzyme or mRNA or guide RNA delivered using
  • nanoparticles or lipid envelopes Discussion herein as to other delivery systems or vectors are because such may he used in conjunction with the nanoparticle aspects of the invention.
  • nanoparticle refers to any particle having a diameter of less than 1000 nm.
  • nanoparticles of the invention have a greatest dimension (e.g., diameter) of 500 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 25 nm and 200 nm.
  • nanoparticles of the invention have a greatest dimension of 100 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 35 nm and 60 nm,
  • Nanoparticles encompassed in the present invention may be provided in different forms, e.g. as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof.
  • Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles).
  • Nanoparticles made of semiconducting material may also he labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present invention.
  • Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self assemble at water/oil interfaces and act as solid surfactants.
  • Su X, Fricke J, Kavanagh DG, Irvine DJ In vitro and in vivo mRNA delivery using lipid-enveioped pH-responsive polymer nanoparticles" Moi. Pharm. 2011 Jun 6;8(3):774-87. doi: 10.1021/mpl00390v/. Epub 201 1 Apr 1) describes
  • PBAE poly(B-amino ester)
  • the pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the poiycation core. Such are, therefore, preferred for delivering RNA of the present invention.
  • nanoparticles based on self-assembling bioadhesive polymers are contemplated, which may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, all to the brain.
  • Other embodiments, such as oral absorption and ocular deliver of hydrophobic drugs are also contemplated.
  • the molecular envelope technology involves an engineered polymer envelope which is protected and delivered to the site of the disease (see, e.g., Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et ai. Mol. Pharm., 2012. 9(1): 14-28; Lalatsa, A., et al.
  • mice involve 20g mammals.
  • US patent application 20110293703 relates to lipidoid compounds are also particularly useful in the administration of polynucleotides, which may be applied to deliver the CRISPR Cas system of the present invention.
  • the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a ceil or a subject to form nanoparticles.
  • the agent to be delivered by the particles may be in the form of a gas, liquid, or solid, and the agent may be a polynucleotide, protein, peptide, or small molecule.
  • the aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles.
  • lipid nanoparticles are contemplated.
  • an antitransthyretin small interfering RNA encapsulated in lipid nanoparticles may be applied to the CRISPR Cas system of the present invention.
  • Doses of about 0.01 to about I mg per kg of body weight administered intravenously are contemplated.
  • Medications to reduce the risk of infusion- related reactions are contemplated, such as dexamethasone, acetaminophen, diphenhydramine or cetiiizine, and ranitidine are contemplated.
  • Multiple doses of about 0.3 mg per kilogram every 4 weeks for five doses are also contemplated.
  • US Patent No. 8,709,843 incorporated herein by reference, provides a drug delivery system for targeted delivery of therapeutic agent-containing particles to tissues, ceils, and intracellular compartments.
  • the invention provides targeted particles comprising polymer conjugated to a surfactant, hydrophilic polymer or lipid.
  • the present invention also contemplates delivering the CRISPR-Cas system to one or both lungs.
  • AAV-2-based vectors were originally proposed for CFTR deliveiy to CF airways, other serotypes such as A A V-l , AA V-5, AAV-6, and AAV-9 exhibit improved gene transfer efficiency in a variety of models of the lung epithelium (see, e.g., Li et al, Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009).
  • AAV-1 was demonstrated to be -0100-fold more efficient than AAV-2 and AAV- 5 at transducing human airway epithelial cells in vitro, 5 although AAV-1 transduced murine tracheal airway epithelia in vivo with an efficiency equal to that of AAV-5.
  • AAV- 5 is 50-fold more efficient than AAV-2 at gene delivery to human airway epithelium (HAE) in vitro and significantly more efficient in the mouse lung airway epithelium in vivo
  • AA V-6 has also been shown to be more efficient than AA V-2 in human airway epithelial cells in vitro and murine airways in vivo.8
  • AAV-9 was shown to display 1- greater gene transfer efficiency than AA V-5 in murine nasal and alveolar epithelia in vivo with gene expression detected for over 9 months showing AAV may enable long-tem l gene expression in vivo, a desirable property for a CFTR gene delivery vector.
  • AA V-9 could be readministered to the murine lung with no loss of CFTR expression and minimal immune consequences.
  • CF and non-CF 1-IAE cultures may be inoculated on the apical surface with 100 ⁇ of AA V vectors for hours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009).
  • the MOI may vary from 1 x 103 to 4 x 105 vector genomes/cell, depending on virus concentration and purposes of the experiments.
  • the above cited vectors are contemplated for the deliveiy and/or administration of the invention.
  • Zamora et al. (Am 1 Respir Crit Care Med Vol 183. pp 531—538, 2011) reported an example of the application of an RN A interference therapeutic to the treatment of human infectious disease and also a randomized trial of an antiviral drug in respiratory syncytial vims (RSV)-infected lung transplant recipients.
  • RSV respiratory syncytial vims
  • Zamora et al. performed a randomized, double-blind, placebo controlled trial in LTX recipients with RSV respiratory tract infection. Patients were permitted to receive standard of care for RSV. Aerosolized ALN-RS V0 1 (0.6 mg/kg) or placebo was administered daily for 3 days. This study demonstrates that an RNAi therapeutic targeting RSV can be safely administered to LTX recipients with RSV infection.
  • Three daily doses of ALN-RS V01 did not result in any exacerbation of respiratory tract symptoms or impairment of lung function and did not exhibit any systemic proinflammatory effects, such as induction of cytokines or CRP.
  • Schwank et al. (Ceil Stem Cell, 13 :653— 58, 2013) used CRISPR/Cas9 to correct a defect associated with cystic fibrosis in human stem cells.
  • the team's target was the gene fix an ion channel, cystic fibrosis transmembrane conductor receptor (CFTR).
  • CFTR cystic fibrosis transmembrane conductor receptor
  • a deletion in CFTR causes the protein to misfold in cystic fibrosis patients.
  • CFTR cystic fibrosis transmembrane conductor receptor
  • a method for in vitro, ex vivo, and/or in vivo functional gene silencing comprising administering a composition comprising a nanoparticle formulation comprising one or more components of the CRISPR-Cas system is provided.
  • a method for in vitro, ex vivo, and/or in vivo functional gene silencing comprising administering a nanoparticle formulation comprising one or more components of the CRISPR-Cas system is provided,
  • a method for in vitro, ex vivo, and/or in vivo functional gene silencing in lung cells comprising a gRNA-nanoparticle formulation comprising one or more guide RNAs is provided.
  • Use of PRRS virus as tag to target cell delivery is provided.
  • the gene editing bundle and nanoparticle may be delivered to targeted cells using an inactivated vims.
  • one embodiment of the invention is delivering to cells which express CD163 .
  • a viral coat protein of the PRRSv virus that binds to CD163, for example, could be attached to the gene-editing bundle and nanoparticle.
  • virus as carrier of the gene-editing bundle and nanoparticle complex
  • a DNA or RNA virus could be used to encapsulate the gene-editing bundle and nanoparticle complex.
  • the capsule could, for example, surround Cas9 as a protein and a guide RNA.
  • the Cas9 could be delivered directly as a protein or encoded in DNA which would be expressed in the cell to express the CRISPRs only in the targeted cells.
  • Cas9 could also be delivered as an RNA.
  • One of ordinary skill in the art would understand that there are methods of stabilizing RNA for such deliver ⁇ '.
  • This invention prevents the introduction of edits into the genome and uses nanoparticles which increase solubility in the cell membrane to deliver the gene editing complex directly into the cell, resulting in non-heritable genetic changes which confer disease resistance.
  • the invention comprises a means for reducing infection and/or transmission of the viral diseases in mammals.
  • Gene editing bundles comprising a nuclease and at least one nucleotide directing where the nuclease should act in the genome, are loaded onto
  • the nanoparticles comprise lipids.
  • the gene editing bundle-nanoparticle complex is then injected into the mammal, where it circulates through the bloodstream and passes through cell membranes to enter the cell, where DNA is edited.
  • administration is through injection into a vein.
  • administration is through nasal administration.
  • administration is through nasal administration wherein administration comprises delivery through injection or aerosol .
  • said cells are alveolar macrophages or their progenitor cells.
  • the gene editing complex edits the DNA. This edit results in the alteration of the expression of the targeted gene in these cells.
  • the gene is CD163.
  • Altered gene expression in these cells prevents the transmission of and/or infection of the mammal with the virus.
  • the virus is porcine reproductive and respiratory syndrome vims (PRRSV).
  • PRRSV porcine reproductive and respiratory syndrome vims
  • the mammal is from the genus Sus and species scrofa. Altered gene expression in these cells prevents the transmission of and/or infection of the mammal with the vims.
  • One specific embodiment involves the production of gene editing treatment for the prevention of PRRSv through delivery of a gene editing nuclease complex attached to lipid molecules, which when delivered intravenously, enter the bloodstream and alter expression of the CD 163 gene in alveolar macrophage cells.
  • One embodiment involves using a nanoparticle delivered nuclease to target the CD 163 mRNA in blood cells and/or the PRRS virus RNA, rather than modifying the DNA.
  • type III CRISPR nucleases and any other systems which target RNA could be contemplated.
  • a guide sequence is about or more than about 5, 0, 1 1 , 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide sequence is 10 - 30 nucleotides long. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to foml a CRISPR complex may he provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a guide sequence may he selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • Exemplary target sequences include those that are unique in the target genome.
  • a unique target sequence in a genome may include a Cas9 target site of the form
  • NNNNNNNXGG N is A, G, T, or C; and X can be anything
  • a unique target sequence in a genome may include an S. thermophilus CRISPR Cas9 target site of the form MMMMMMMMMN N NNXXAG AAW where
  • NNNNNNNNNN NNXXAGAAW N is A, G, T, or C; and X can be anything
  • a guide sequence is selected to reduce the degree secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, I %, or fewer of the nucleotides of the guide sequence participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is niFold, as described by Zuker and Stiegler (Nucleic Acids Res.
  • Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. G ruber et a!., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1 151 -62).
  • a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence.
  • degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may he determined by any suitable alignment algorithm, and may further account for secondary structures, such as self- complementarity within either the tracr sequence or tracr mate sequence.
  • the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the transcript or transcribed polynucleotide sequence has at least two or more hairpins.
  • the transcript has two, three, four or five hairpins.
  • the transcript has at most five hairpins. In a hairpin structure the portion of the sequence 5' of the final "N" and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3' of the loop corresponds to the tracr sequence.
  • Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. For example, for the guide sequence targeting 5' ⁇
  • CRISPR enzyme nickase mRNA for example S. pyogenes Cas9 with the D10A mutation
  • CRISPR enzyme nickase mRNA can be delivered with a pair of guide RNAs targeting a site of interest. Multiple methods exist to increase specificity to the targeting guideRNA.
  • One such approach is a method comprising: (1) contacting a selected target nucleic acid comprising a region to be modified with (a) an agent that suppresses alternative end- joining (alt- EJ)/micro homology mediated end-joining (MMEJ), thereby favoring nonhomologous end-joining (NHEJ); and (b) a DNA binding molecule that targets the selected target nucleic acid; and (2) producing one or more double-strand breaks in the targeted region using a programmable endonuclease, thereby triggering DNA repair pathways to repair the breaks, whereby repair of the cleaved target nucleic acid proceeds substantially by MMEJ and is done in the absence of a donor polynucleotide, thereby modulating the DNA repair outcome, as taught in PCT/US201 7/018679, incorporated by reference in its entirety herein.
  • CRISPR system comprises: contacting a target nucleic acid molecule having a target sequence with: a single polynucleotide comprising a targeting region comprising deoxyribonucleic acid (DNA) and configured to hybridize with a target sequence in a nucleic acid, an activating region adjacent to said targeting region comprising a ribonucleic acid (RNA); and a site-directed polypeptide, wherein the single polynucleotide forms a complex with the site-directed polypeptide and wherein said target nucleic acid molecule is cleaved or edited at the target sequence more preferentially than at other sequences in the target nucleic acid, thereby reducing off-target modification.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the target nucleic acid is DNA, in some embodiments the target nucleic acid is RNA, in some embodiments the target nucleic acid is a mixture of RNA and DNA.
  • the activating region is downstream of the targeting region. In some embodiments, the activating region is upstream of the targeting region.
  • the site-directed polypeptide is a Cas9 protein. In some embodiments, the site-directed polypeptide is a Cpf 1 protein.
  • the activating region comprises a structure selected from the group consisting of a lower stem, a bulge, an upper stem, a nexus, and a hairpin. In some embodiments, the activating region comprises a stem loop structure.
  • the activating region interacts with the site-directed polypeptide.
  • the activating region comprises a mixture of DNA and RNA.
  • the targeting region comprises a mixture of DNA and RNA.
  • said targeting region is free of uracil.
  • the method further includes providing a donor polynucleotide.
  • this hybrid guideRNA is not limited to Type II Cas systems, but also applies to any polynucleotide-directed endonuclease including but not limited to Cpfl, Type 1 , Type III, Type IV, Cmsl, and Csml (disclosed in U.S. Patent No. 9,896,696, hereby incorporated by reference in its entirety).
  • CRISPR enzyme or CRISPR enzyme mRNA or CRISPR guide RNA or RNA(s) can be delivered separately; and advantageously at least one of these is delivered via a nanoparticle complex.
  • CRISPR enzyme mRNA can be delivered prior to the guide RNA to give time for CRISPR enzyme to be expressed.
  • CRISPR enzyme mRNA might be administered 1-12 hours (preferably around 2-6 hours) prior to the administration of guide RNA.
  • CRISPR enzyme mRNA and guide R A can be administered together.
  • a second booster dose of guide RNA can be administered 1 -12 hours (preferably around 2-6 hours) after the initial administration of CRISPR enzyme mRNA + guide RNA. Additional administrations of CRISPR enzyme mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome modification.
  • An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide.
  • the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
  • this invention provides a method of cleaving a target polynucleotide. The method comprises modifying a target
  • the CRISPR complex of the invention when introduced into a cell, creates a break (e.g., a single or a double strand break) in the genome sequence.
  • a break e.g., a single or a double strand break
  • the method can be used to cleave a disease gene in a cell.
  • the break created by the CRISPR complex can be repaired by a repair processes such as the error prone non-homologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR).
  • NHEJ error prone non-homologous end joining
  • HDR high fidelity homology-directed repair
  • a donor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PGR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • the exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene).
  • the sequence for integration may be a sequence endogenous or exogenous to the ceil.
  • sequences to be integrated examples include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA).
  • sequence for integration may be operably linked to an appropriate control sequence or sequences.
  • sequence to be integrated may provide a regulator ⁇ ' function.
  • the upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide.
  • a target polynucleotide can be inactivated to affect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does.
  • the inactivation occurs at the CD 163 locus.
  • the target polynucleotide is CD 163 mRNA.
  • One example of inactivating the CD 163 locus is taught in Prather et. al., U.S. Patent No. 10,080,353, hereby incorporated by reference in its entirety.
  • site-specific integration of an exogenous nucleic acid at a GDI 63 locus may be accomplished by any technique known to those of skill in the art.
  • integration of an exogenous nucleic acid at a CD 163 locus comprises contacting a cell (e.g., an isolated cell or a cell in a tissue or organism) with a nucleic acid molecule comprising the exogenous nucleic acid.
  • a nucleic acid molecule may comprise nucleotide sequences flanking the exogenous nucleic acid that facilitate homologous recombination between the nucleic acid molecule and at least one GDI 63 locus.
  • the nucleotide sequences flanking the exogenous nucleic acid that facilitate homologous recombination may be
  • homologous recombination may be complementary to previously integrated exogenous nucleotides.
  • a plurality of exogenous nucleic acids may be integrated at one CD 163 locus, such as in gene stacking.
  • Integration of a nucleic acid at a GDI 63 locus may be facilitated (e.g., catalyzed) in some embodiments by endogenous cellular machinery of a host cell, such as, for example and without limitation, endogenous DNA and endogenous recombinase enzymes.
  • integration of a nucleic acid at a GDI 63 locus may be facilitated by one or more factors (e.g., polypeptides) that are provided to a host cell.
  • factors e.g., polypeptides
  • nuclease(s), recombinase(s), and/or ligase polypeptides may be provided (either independently or as part of a chimeric polypeptide) by contacting the polypeptides with the host cell, or by expressing the polypeptides within the host cell.
  • a nucleic acid comprising a nucleotide sequence encoding at least one nuclease, recombinase, and/or ligase polypeptide may be introduced into the host cell, either concurrently or sequentially with a nucleic acid to he integrated site-specifically at a CD163 locus, wherein the at least one nuclease, recombinase, and/or ligase polypeptide is expressed from the nucleotide sequence in the host cell.
  • site-specific integration may be accomplished by utilizing factors that are capable of recognizing and binding to particular nucleotide sequences, for example, in the genome of a host organism.
  • proteins comprise polypeptide domains that are capable of recognizing and binding to DNA in a site-specific manner.
  • a DNA sequence that is recognized by a DNA-binding polypeptide may be referred to as a "target" sequence.
  • Polypeptide domains that are capable of recognizing and binding to DNA in a site-specific manner generally fold correctly and function independently to bind DNA in a site-specific manner, even when expressed in a polypeptide other than the protein from which the domain was originally isolated.
  • target sequences for recognition and binding by DNA-binding polypeptides are generally able to be recognized and bound by such polypeptides, even when present in large DNA structures (e.g., a chromosome), particularly when the site where the target sequence is located is one known to be accessible to soluble cellular proteins (e.g., a gene).
  • DNA-binding polypeptides identified from proteins that exist in nature typically bind to a discrete nucleotide sequence or motif (e.g., a consensus recognition sequence), methods exist and are known in the art for modifying many such DN A-binding polypeptides to recognize a different nucleotide sequence or motif.
  • a discrete nucleotide sequence or motif e.g., a consensus recognition sequence
  • DNA-binding polypeptides include, for example and without limitation: zinc finger DNA-binding domains; leucine zippers; UP A DNA-binding domains; GAL4; TAL; LexA; a Tet repressor; LacR; and a steroid hormone receptor.
  • a DNA-binding polypeptide is a zinc finger.
  • Canonical Cys2His2 (as well as non-canonical Cys3His) zinc finger polypeptides bind DNA by inserting an a-helix into the major groove of the target DNA double helix.
  • Recognition of DNA by a zinc finger is modular; each finger contacts primarily three consecutive base pairs in the target, and a few key residues in the polypeptide mediate recognition.
  • the DNA-binding specificity of the targeting endonuclease may be further increased (and hence the specificity of any gene regulatory effects conferred thereby may also be increased).
  • one or more zinc finger DNA-binding polypeptides may be engineered and utilized such that a targeting endonuclease introduced into a host ceil interacts with a DNA sequence that is unique within the genome of the host cell.
  • the zinc finger protein is non-natural ly occurring in that it is engineered to bind to a target site of choice.
  • a target site of choice See, for example, See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135- 141 ; Pabo et al . (2001 ) Ann, Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637, Choo et al. (2000) Curr. Opin. Struct. Biol.
  • An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein.
  • Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261 , incorporated by reference herein in their entireties.
  • zinc finger domains and/or multi-fmgered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length.
  • the proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.
  • zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length.
  • the proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.
  • a DNA-binding polypeptide is a D A-binding domain from GAL4.
  • GAL4 is a modular transact! vator in Saccharomyces cerevisiae, but it also operates as a transact! vator in many other organisms. See, e.g., Sadowski et al. (1988) Nature 335:563-4.
  • the expression of genes encoding enzymes of the galactose metabolic pathway in S. cerevisiae is stringently regulated by the available carbon source. Johnston (1987) Microbiol. Rev. 51 :458-76. Transcriptional control of these metabolic enzymes is mediated by the interaction between the positive regulatory protein, GAL4, and a 17 bp symmetrical DNA sequence to which GAL4 specifically binds (the UAS).
  • Native GAL4 consists of 881 amino acid residues, with a molecular weight of 99 kDa. GAL4 comprises functionally autonomous domains, the combined activities of which account for activity of GAL4 in vivo. Ma and Ptashne (1987) Cell 48:847-53); Brent and Ptashne (1985) Cell 43(3 Pt 2 : ⁇ : 729-36. The N-terminal 65 amino acids of GAL4 comprise the GAL4 DNA-binding domain. Keegan et al. (1986) Science 231 :699-704; Johnston (1987) Nature 328:353-5. Sequence-specific binding requires the presence of a divalent cation coordinated by 6 Cys residues present in the DNA binding domain.
  • the coordinated cation-containing domain interacts with and recognizes a conserved CCG triplet at each end of the 1 7 bp UAS via direct contacts with the major groove of the DNA helix.
  • the DNA-binding function of the protein positions C-terminal transcriptional activating domains in the vicinity of the promoter, such that the activating domains can direct transcription.
  • Additional DNA-binding polypeptides that may be utilized in certain embodiments include, for example and without limitation, a binding sequence from a AVRBS3-inducible gene; a consensus binding sequence from a AVRBS3 -inducible gene or synthetic binding sequence engineered therefrom (e.g., UPA DNA-binding domain); TAL; LexA (see, e.g.. Brent & Ptashne (1985), supra); LacR (see, e.g., Labow et al. (1990) Mol. Cell. Biol. 10:3343-56; Bairn et al. (1991) Proc. Natl. Acad. Sci.
  • the DNA-binding domain of one or more of the nucleases used in the methods and compositions described herein comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA. binding domain. See, e.g., U.S. Patent Publication No. 201 10301073, incorporated by reference in its entirety herein.
  • the DNA-binding domain of one or more of the nucleases used in the methods and compositions described herein comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain. See, e.g., U.S. Patent Publication No. 201 10301073, incorporated by reference in its entirety herein.
  • the nuclease comprises a CRISPR/Cas system.
  • the CRISPR (clustered regularly interspaced short palindromic repeats) locus which encodes RNA components of the system
  • the Cas (CRISPR-associated) locus which encodes proteins (Jansen et al., 2002. Mol. Microbiol. 43 : 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al, 2006. Biol. Direct 1 : 7; Haft et al., 2005. PLoS Comput. Biol.
  • CRISPR loci in microbial hosts contain a combination of Cas genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.
  • the Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps.
  • the mature crRNA: tracrRNA complex directs Cas9 to the target DNA via Wastson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition.
  • Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. Activity of the
  • CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the foreign nucleic acid.
  • a process called 'adaptation' a process called 'adaptation'
  • expression of the relevant proteins as well as expression and processing of the array
  • RNA-mediated interference with the foreign nucleic acid RNA-mediated interference with the foreign nucleic acid.
  • CRISPR/Cas system serve roles in functions such as insertion of the foreign DNA etc.
  • Cas protein may be a "functional derivative” of a naturally occurring Cas protein.
  • a “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide.
  • “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a
  • a biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments.
  • the term "derivative" encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof.
  • Cas protein which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures.
  • the cell may be a cell that naturally produces Cas protein, or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas that is same or different from the endogenous Cas, In some case, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein.
  • a DNA-binding polypeptide specifically recognizes and binds to a target nucleotide sequence comprised within a genomic nucleic acid of a host organism. Any number of discrete instances of the target nucleotide sequence may be found in the host genome in some examples.
  • the target nucleotide sequence may be rare within the genome of the organism (e.g., fewer than about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 copy(ies) of the target sequence may exist in the genome).
  • the target nucleotide sequence may be located at a unique site within the genome of the organism.
  • Target nucleotide sequences may be, for example and without limitation, randomly dispersed throughout the genome with respect to one another; located in different linkage groups in the genome; located in the same linkage group; located on different chromosomes; located on the same chromosome; located in the genome at sites that are expressed under similar conditions in the organism (e.g., under the control of the same, or substantially functionally identical, regulatory factors); and located closely to one another in the genome (e.g., target sequences may be comprised within nucleic acids integrated as concatemers at genomic loci).
  • a DNA-binding polypeptide that specifically recognizes and binds to a target nucleotide sequence may be comprised within a chimeric polypeptide, so as to confer specific binding to the target sequence upon the chimeric polypeptide.
  • a chimeric polypeptide may comprise, for example and without limitation, nuclease, recombinase, and/or ligase polypeptides, as these polypeptides are described above.
  • Chimeric polypeptides comprising a DNA- binding polypeptide and a nuclease, recombinase, and/or ligase polypeptide may also comprise other functional polypeptide motifs and/or domains, such as for example and without limitation: a spacer sequence positioned between the functional polypeptides in the chimeric protein; a leader peptide; a peptide that targets the fusion protein to an organelle (e.g., the nucleus); polypeptides that are cleaved by a cellular enzyme, peptide tags (e.g., Myc, His, etc.); and other amino acid sequences that do not interfere with the function of the chimeric polypeptide,
  • Functional polypeptides e.g., DNA-binding polypeptides and nuclease polypeptides
  • a chimeric polypeptide may be operatively linked.
  • functional polypeptides of a chimeric polypeptide may be operatively linked by their expression from a single polynucleotide encoding at least the functional polypeptides li gated to each other in-frame, so as to create a chimeric gene encoding a chimeric protein.
  • the functional polypeptides of a chimeric polypeptide may be operatively linked by other means, such as by cross-linkage of independently expressed polypeptides,
  • a DNA-binding polypeptide, or guide RNA that specifically recognizes and binds to a target nucleotide sequence may be comprised within a natural isolated protein (or mutant thereof), wherein the natural isolated protein or mutant thereof also comprises a nuclease polypeptide (and may also comprise a recombinase and/or ligase polypeptide).
  • isolated proteins include TALENs, recombinases (e.g., Cre, Hin, Tre, and FLP recombinase), RNA- guided CRISPR/Cas9, and meganucleases.
  • targeting endonuclease refers to natural or engineered isolated proteins and mutants thereof that comprise a DNA-binding polypeptide or guide RNA and a nuclease polypeptide, as well as to chimeric polypeptides comprising a DNA- binding polypeptide or guide RNA and a nuclease.
  • Any targeting endonuclease comprising a DNA-binding polypeptide or guide RNA that specifically recognizes and binds to a target nucleotide sequence comprised within a CD 163 locus (e.g., either because the target sequence is comprised within the native sequence at the locus, or because the target sequence has been introduced into the locus, for example, by recombination) may be utilized in certain embodiments.
  • chimeric polypeptides that may be useful in particular embodiments of the invention include, without limitation, combinations of the following polypeptides: zinc finger DNA-binding polypeptides; a Fokl nuclease polypeptide; TALE domains; leucine zippers; transcription factor DNA-binding motifs; and DNA recognition and/or cleavage domains isolated from, for example and without limitation, a TALEN, a recombinase (e.g., Cre, Hin, RecA, Tre, and FLP recombinases), RNA-guided CRISPR/Cas9, a meganuclease; and others known to those in the art.
  • TALEN a recombinase
  • Cre Cre, Hin, RecA, Tre, and FLP recombinases
  • CRISPR/Cas9 a meganuclease
  • Chimeric polypeptides may be engineered by methods known to those of skill in the art to alter the recognition sequence of a DNA-binding polypeptide comprised within the chimeric polypeptide, so as to target the chimeric polypeptide to a particular nucleotide sequence of interest.
  • the chimeric polypeptide comprises a DNA- binding domain (e.g., zinc finger, TAL-effector domain, etc.) and a nuclease (cleavage) domain.
  • the cleavage domain may be heterologous to the DNA-binding domain, for example a zinc finger DNA-binding domain and a cleavage domain from a nuclease or a TALEN DNA- binding domain and a cleavage domain, or meganuclease DNA- binding domain and cleavage domain from a different nuclease.
  • Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary
  • endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass., and Belfort et al. (1997) Nucleic Acids Res, 25:3379-3388. Additional enzymes which cleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease, see also Linn et al . (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.
  • a cleavage half-domain can be derived from any nuclease or portion i hereof, as set forth above, that requires dimerization for cleavage activity.
  • two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains.
  • a single protein comprising two cleavage half-domains can be used.
  • the two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof).
  • the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation to each other that allows the cleavage half- domains to form a functional cleavage domain, e.g., by dimerizing.
  • the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides.
  • any integral number of nucleotides, or nucleotide pairs can intervene between two target sites (e.g., from 2 to 50 nucleotide pairs or more).
  • the site of cleavage lies between the target sites,
  • Restriction endonucleases are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding, for example, such that one or more exogenous sequences (donors/transgenes) are integrated at or near the binding (target) sites.
  • Certain restriction enzymes e.g., Type IIS
  • Fok I catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other.
  • fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
  • An exemplary Type IIS restriction enzyme whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc, Natl. Acad. Sci. USA 95 : 10,570- 10,575.
  • the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain.
  • two fusion proteins, each comprising a Fokl cleavage half-domain can be used to reconstitute a catalytically active cleavage domain.
  • a single polypeptide molecule containing a DNA binding domain and two Fok I cleavage half-domains can also be used.
  • a cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.
  • Exemplary Type IIS restriction enzymes are described in U.S. Patent Publication No. 20070134796, incorporated herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31 :418-420.
  • the cleavage domain comprises one or more engineered cleavage half-domain (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 20050064474; 20060188987 and 20080131962, the disclosures of all of which are incorporated by reference in their entireties herein.
  • engineered cleavage half-domain also referred to as dimerization domain mutants
  • nucleases may be assembled in vivo at the nucleic acid target site using so-called "split-enzyme” technology (see e.g. U.S. Patent Publication No. 20090068164).
  • split-enzyme e.g. U.S. Patent Publication No. 20090068164.
  • Components of such split enzymes may be expressed either on separate expression constructs or can be linked in one open reading frame where the individual components are separated, for example, by a self-cleaving 2A peptide or IRES sequence.
  • Components may be individual zinc finger binding domains or domains of a meganuclease nucleic acid binding domain.
  • a chimeric polypeptide is a custom-designed zinc finger nuclease (ZFN) that may be designed to deliver a targeted site-specific double-strand DNA break into which an exogenous nucleic acid, or donor DNA, may be integrated (See co-owned US Patent publication 20100257638, incorporated by reference herein).
  • ZFNs are chimeric polypeptides containing a non-specific cleavage domain from a restriction endonuclease (for example, Fokl) and a zinc finger DNA- binding domain polypeptide. See, e.g., Fluang et al. (1996) J. Protein Chem. 15:481 -9; Kim et al. (1997a) Proc. Natl.
  • the ZFNs comprise non-canonical zinc finger DNA binding domains (see US Patent publication 20080182332, incorporated by reference herein).
  • the Fokl restriction endonuclease must dimerize via the nuclease domain in order to cleave DNA and introduce a double-strand break.
  • ZFNs containing a nuclease domain from such an endonuclease also require dimerization of the nuclease domain in order to cleave target DNA.
  • Dimerization of the ZFN can be facilitated by two adjacent, oppositely oriented DNA -binding sites. Id.
  • a method for the site-specific integration of an exogenous nucleic acid into at least one CD 163 locus of a host comprises introducing into a cell of the host a ZFN, wherein the ZFN recognizes and binds to a target nucleotide sequence, wherein the target nucleotide sequence is comprised within at least one CD 163 locus of the host.
  • the target nucleotide sequence is not comprised within the genome of the host at any other position than the at least one CD163 locus.
  • a DNA-binding polypeptide of the ZFN may be engineered to recognize and bind to a target nucleotide sequence identified within the at least one CD 163 locus (e.g., by sequencing the CD 163 locus),
  • a method for the site- specific integration of an exogenous nucleic acid into at least one CD163 performance locus of a host that comprises introducing into a cell of the host a ZFN may also comprise introducing into the cell an exogenous nucleic acid, wherein recombination of the exogenous nucleic acid into a nucleic acid of the host comprising the at least one GDI 63 locus is facilitated by site- specific recognition and binding of the ZFN to the target sequence (and subsequent cleavage of the nucleic acid comprising the CD 163 locus).
  • Embodiments of the invention may include one or more nucleic acids selected from the group consisting of: an exogenous nucleic acid for site-specific integration in at least one CD 163 locus, for example and without limitation, an ORF; a nucleic acid comprising a nucleotide sequence encoding a targeting endonuclease; and a vector comprising at least one of either or both of the foregoing.
  • nucleic acids for use in some embodiments include nucleotide sequences encoding a polypeptide, structural nucleotide sequences, and/or DNA-binding polypeptide recognition and binding sites.
  • donor sequence also called a "donor sequence” or “donor”
  • donor sequence is typically not identical to the genomic sequence where it is placed.
  • a donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest.
  • donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
  • a donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
  • the donor polynucleotide can be DNA or R A, single-stranded or double-stranded and can be introduced into a ceil in linear or circular form, as a hybrid molecule as well as a DNA-RNA hybrid donor. See e.g., U.S. Patent Publication Nos. 20100047805, 201 10281361 , 201 10207221 and U.S. application Ser. No. 13/889,162.
  • the ends of the donor sequence can be protected (e.g. from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and 0-methyl ribose or deoxyribose residues,
  • a polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
  • donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).
  • viruses e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)
  • the donor is generally integrated so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is integrated (e.g., CD 163).
  • the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.
  • exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or poiyadenylation signals.
  • Exogenous nucleic acids that may be integrated in a site-specific manner into at least one CD 163 locus, so as to modify the CD163 locus, in embodiments include, for example and without limitation, nucleic acids comprising a nucleotide sequence encoding a polypeptide of interest; nucleic acids comprising an agronomic gene; nucleic acids comprising a nucleotide sequence encoding an R Ai molecule; or nucleic acids that disrupt the GDI 63 gene.
  • an exogenous nucleic acid is integrated at a CD 163 locus, so as to modify the CD 163 locus, wherein the nucleic acid comprises a nucleotide sequence encoding a polypeptide of interest, such that the nucleotide sequence is expressed in the host from the CD 163 locus.
  • the polypeptide of interest e.g., a foreign protein
  • the polypeptide of interest may be extracted from the host cell, tissue, or biomass.
  • a nucleotide sequence encoding a targeting endonuclease may be engineered by manipulation (e.g., ligation) of native nucleotide sequences encoding polypeptides comprised within the targeting endonuclease.
  • the nucleotide sequence of a gene encoding a protein comprising a DNA- binding polypeptide may be inspected to identify the nucleotide sequence of the gene that corresponds to the DNA-binding polypeptide, and that nucleotide sequence may be used as an element of a nucleotide sequence encoding a targeting endonuclease comprising the DNA-binding polypeptide.
  • the amino acid sequence of a targeting endonuclease may be used to deduce a nucleotide sequence encoding the targeting endonuclease, for example, according to the degeneracy of the genetic code.
  • nucleic acid molecules comprising a nucleotide sequence encoding a targeting endonuclease
  • the last codon of a first polynucleotide sequence encoding a nuclease polypeptide, and the first codon of a second polynucleotide sequence encoding a DNA-binding polypeptide may be separated by any number of nucleotide triplets, e.g., without coding for an intron or a "STOP.”
  • the last codon of a nucleotide sequence encoding a first polynucleotide sequence encoding a DNA-binding polypeptide, and the first codon of a second polynucleotide sequence encoding a nuclease polypeptide may he separated by any number of nucleotide triplets.
  • the last codon of the last (i.e., most 3' in the nucleic acid sequence) of a first polynucleotide sequence encoding a nuclease polypeptide, and a second polynucleotide sequence encoding a DNA-binding polypeptide may be fused in phase-register with the first codon of a further polynucleotide coding sequence directly contiguous thereto, or separated therefrom by no more than a short peptide sequence, such as that encoded by a synthetic nucleotide linker (e.g., a nucleotide linker that may have been used to achieve the fusion).
  • a synthetic nucleotide linker e.g., a nucleotide linker that may have been used to achieve the fusion.
  • polynucleotide sequences include, for example and without limitation, tags, targeting peptides, and enzymatic cleavage sites.
  • tags for example and without limitation, tags, targeting peptides, and enzymatic cleavage sites.
  • polynucleotide sequences may be fused in phase -register with the last codon of a further polynucleotide coding sequence directly contiguous thereto or separated therefrom by no more than a short peptide sequence.
  • a sequence separating polynucleotide sequences encoding functional polypeptides in a targeting endonuclea.se may, for example, consist of any sequence, such that the amino acid sequence encoded is not likely to significantly alter the translation of the targeting endonuclease. Due to the autonomous nature of known nuclease polypeptides and known DNA-binding polypeptides, intervening sequences will not in examples interfere with the respective functions of these structures.
  • a CD 163 locus is a CD 163 homologue (e.g., an ortholog or a paralog) that comprises a nucleotide sequence that is at least about 85% identical to CD163.
  • a CD163 homologue may comprise a nucleotide sequence that is, for example and without limitation: at least 80%; at least 85%; at least about 90%; at least about 91%; at least about 92%; at least about 93%; at least about 94%; at least about 95%, at least about 96%; at least about 97%; at least about 98%; at least about 99%; at least about 99.5%; 99.6%, 99.7%, 99.8% and/or at least about 99.9% identical to about 20 contiguous nucleotides of a nucleotide sequence for CD 163.
  • CD 163 nucleotide sequence can be modified as described as in US 2017/0035035 Al to Prather et al., which is expressly incorporated herein in its entirety, including the figures, tables, and sequences.

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Abstract

L'invention concerne un moyen pour réduire l'infection et/ou la transmission de maladies virales chez les mammifères. Des complexes d'édition de gènes, comprenant une nucléase et au moins un nucléotide ciblant l'endroit où la nucléase devrait agir dans le génome, sont chargés sur des nanoparticules. Le complexe ensemble éditions de gènes-nanoparticule est ensuite administré au mammifère et traverse les membranes cellulaires pour pénétrer dans la cellule, où l'ADN est édité. Une fois à l'intérieur des cellules, le complexe d'édition de gènes édite l'ADN. L'édition engendre une altération de l'expression du gène ciblé dans ces cellules. La présente invention utilise des nanoparticules qui augmentent la solubilité dans la membrane cellulaire pour pouvoir administrer le complexe d'édition de gènes directement dans la cellule, avec pour résultat des changements génétiques non héritables qui confèrent une résistance aux maladies.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014150624A1 (fr) * 2013-03-14 2014-09-25 Caribou Biosciences, Inc. Compositions et procédés pour des acides nucléiques à ciblage d'acide nucléique
WO2015011483A1 (fr) * 2013-07-24 2015-01-29 The University Court Of The University Of Edinburgh Domaine 5 de cd163 destiné à être utilisé dans des compositions antivirales dirigées contre prrs, et animaux transgeniques
WO2017023337A1 (fr) * 2015-08-06 2017-02-09 The Curators Of The University Of Missouri Animaux résistants à des agents pathogènes ayant des gènes cd163 modifiés
WO2017210666A2 (fr) * 2016-06-03 2017-12-07 Stemgenics, Inc. Nanoparticules fonctionnalisées pour l'administration intracellulaire de molécules biologiquement actives et leurs procédés de fabrication et d'utilisation

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Publication number Priority date Publication date Assignee Title
WO2014150624A1 (fr) * 2013-03-14 2014-09-25 Caribou Biosciences, Inc. Compositions et procédés pour des acides nucléiques à ciblage d'acide nucléique
WO2015011483A1 (fr) * 2013-07-24 2015-01-29 The University Court Of The University Of Edinburgh Domaine 5 de cd163 destiné à être utilisé dans des compositions antivirales dirigées contre prrs, et animaux transgeniques
WO2017023337A1 (fr) * 2015-08-06 2017-02-09 The Curators Of The University Of Missouri Animaux résistants à des agents pathogènes ayant des gènes cd163 modifiés
WO2017210666A2 (fr) * 2016-06-03 2017-12-07 Stemgenics, Inc. Nanoparticules fonctionnalisées pour l'administration intracellulaire de molécules biologiquement actives et leurs procédés de fabrication et d'utilisation

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