Cold-tolerant crispr / cas systems

Abstract

Provided herein are non-naturally occurring CRISPR / Cas system for modifying a genome at a cold temperature, comprising a cold-tolerant Cas effector or functional fragment thereof and a guide RNA. Also provided are methods of modifying a genome of a cell at a cold temperature.

Description

TITLE: COLD-TOLERANT CRISPR / CAS SYSTEMSCROSS REFERENCE:

[0001] This PCT application claims the benefit to U.S. Provisional Application No. 63 / 555,774, filed February 20, 2024, the content of which is incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING:

[0002] This disclosure contains a Sequence Listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The accompanying Sequence Listing file named “062649-502001 WO_Sequence Listing ST26.xml” was created on February 18, 2025 and is 69,632 bytesFIELD:

[0003] The present disclosure generally relates to CRISPR / Cas systems, more specifically, to CRISPR / Cas systems that can function at low temperatures.BACKGROUND:

[0004] Genome editing in cold environments, particularly below 15°C, has been unexplored, hence lacking optimized editing tools. There remains a need for novel gene editing systems.SUMMARY:

[0005] In one aspect, provided herein is a non-naturally occurring CRISPR / Cas system for modifying a genome of a cell at a cold temperature, comprising a cold-tolerant Cas effector or functional fragment thereof and a guide RNA, wherein the guide RNA targets the Cas effector to a target site in the genome.

[0006] In some embodiments, the cold temperature comprises a temperature below about 20QC, below about 15QC, below about 10QC, below about 5QC, below about 0QC, or below about -5QC.

[0007] In some embodiments, the Cas effector of a Cas9.

[0008] In some embodiments, the cold-tolerant Cas effector is derived from a psychrophile.

[0009] In some embodiments, the psychrophile is a Psychroflexus. In some embodiments, the Psychroflexus is Psychroflexus torquis.

[0010] In some embodiments, the cold-tolerant Cas effector is Psychroflexus torquis Cas9 (PtoCas9).

[0011] In some embodiments, the PtoCas9 comprises an amino acid sequence of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 1 .

[0012] In some embodiments, the cold-tolerant Cas effector is derived from Vibrio natriegens.

[0013] In some embodiments, the cold-tolerant Cas effector is Vibrio natriegens Cas9 (VnaCas9).

[0014] In some embodiments, the VnaCas9 comprises an amino acid sequence of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 6.

[0015] In some embodiments, the guide RNA targets PtoCas9 to a target site in the genome.

[0016] In some embodiments, the guide RNA comprises a sequence of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 25.

[0017] In some embodiments, the guide RNA targets VnaCas9 to a target site in the genome.

[0018] In some embodiments, the guide RNA comprises a sequence of at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 26.

[0019] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human animal cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is an insect cell.

[0020] In some embodiments, the cell is a cell of a tissue or organ. In some embodiments, the tissue or organ is a human tissue or organ. In some embodiments, the tissue or organ is a non-human animal tissue or organ. In some embodiments, the tissue or organ is a tissue or organ for transplantation.

[0021] In another aspect, provided herein are expression cassettes comprising a nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system, operably linked to a promoter.

[0022] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 2.

[0023] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 3.

[0024] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 4.

[0025] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 5.

[0026] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85%sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 7.

[0027] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 8.

[0028] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 9.

[0029] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 10.

[0030] In another aspect, provided herein are expression cassettes comprising a nucleic acid encoding the guide RNA of the CRISPR / Cas system, operably linked to a promoter.

[0031] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 11 .

[0032] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 12.

[0033] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 13.

[0034] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 14.

[0035] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 15.

[0036] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 16.

[0037] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 17.

[0038] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 18.

[0039] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 19.

[0040] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 20.

[0041] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 21 .

[0042] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 22.

[0043] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 23.

[0044] In some embodiments, the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 24.

[0045] In another aspect, provided herein are vectors comprising one or more of the expression cassettes disclosed herein.

[0046] In some embodiments, the vector a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno- associated virus (AAV) vector, a lentiviral vector, or a retroviral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the vector is an adenoviral vector.

[0047] In some embodiments, the vector a non-viral vector.

[0048] In another aspect, provided herein are methods of modifying a genome of a cell at a cold temperature, comprising contacting the cell with the CRISPR / Cas system disclosed herein, thereby modifying the genome at the cold temperature.

[0049] In another aspect, provided herein are methods of modifying a genome of a cell at a cold temperature, comprising contacting the cell with a CRISPR / Cas system comprising a cold-tolerant Cas effector or functional fragment thereof, and a guide RNA,wherein the guide RNA targets the Cas effector or functional fragment thereof to a target site in the genome, thereby modifying the genome at the cold temperature.

[0050] In some embodiments, the cell is a cell of a tissue or an organ. In some embodiments, the tissue or organ is a tissue or organ for transplantation. In some embodiments, the tissue or organ is at a hypothermic state.

[0051] In some embodiments, the hypothermic state comprises a temperature of between about 0QC and about 10QC, or between about 4QC and about 10QC.

[0052] In some embodiments, the hypothermic state comprises sub-zero temperatures.

[0053] In some embodiments, the hypothermic state comprises a temperature of between about -10QC and about 10QC, between about -8QC and about 10QC, between about -6QC and about 10QC, between about -4QC and about 10QC or between about -2QC and about 10QC.

[0054] In some embodiments, contacting the cell with the CRISPR / Cas system comprises performing ex vivo organ perfusion (EVOP).

[0055] In some embodiments, the method further comprises preserving the tissue or organ at a first temperature before performing the EVOP.

[0056] In some embodiments, the first temperature is about 10QC.

[0057] In some embodiments, the method further comprises preserving the tissue or organ at a second temperature before performing the EVOP.

[0058] In some embodiments, the second temperature is about 4QC or about 10QC.

[0059] In some embodiments, the tissue or organ is the lung.

[0060] In some embodiments, the EVOP is ex vivo lung perfusion (EVLP).

[0061] In some embodiments, modifying a genome of a cell results in a change in an expression level of a gene in the genome.

[0062] In some embodiments, the target site comprises an inflammation-regulating and / or immune-regulating gene. In some embodiments, the target site comprises a geneencoding a cytokine. In some embodiments, the target site comprises a gene encoding IL-10.

[0063] Other features and advantages of the present disclosure will become apparent from the following detailed description. The detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only. These detailed description and specific examples are provided for the purposes of explanation, and not limitation, of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS:

[0064] Figs. 1 A-1 C show the impact of low temperature on DNA cleavage activity by SpyCas9. (A) Protein (left) and RNA (right) gel electrophoresis of the purified SpyCas9- gRNA complex. Arrows indicate SpyCas9 protein and gRNA. (B) In vitro cleavage assay using purified SpyCas9 RNP. The 3-kb PCR amplicons (open arrowheads) containing the target locus were designed to generate 2-kb and 1 -kb DNA fragments (arrowheads) after cleavage. The DNA substrate and RNP were incubated at 37QC for 30 min for cleavage reaction. The reaction was stopped by adding EDTA. The DNA fragments were analyzed in agarose gel electrophoresis. (C) The cleavage efficiency at different temperatures were analyzed through quantification of DNA fragments using bioanalyzer.

[0065] Figs. 2A-2D are schematic drawings of a representation of the CRISPR / Cas system according to an embodiment of the present disclosure. (A) The schematic of CRISPR locus in the Psychroflexus torquis genome. A Cas9 gene, which is 4530 bp in size, and CRISPR array, which contain 24 sets of spacer and repeat sequences, were identified. The putative tracrRNA was identified in the upstream of the Cas9 gene by searching anti-repeat sequence. (B) A schematic of computationally predicted crRNA and tracrRNA duplex. (C) A predicted secondary structure of a designed sgRNA. (D) WebLogo image of predicted PAM.

[0066] Figs 3A-3B are schematic drawings of an example approach of developing psychrophilic editing enzymes for therapeutic use.

[0067] Figs. 4A-4B are schematic drawings of an example method of modulating gene expression in lungs according to one embodiment.DETAILED DESCRIPTION OF THE DISCLOSURE:

[0068] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0069] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0070] For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.I. Compositions

[0071] Disclosed herein are CRISPR / Cas systems. The CRISPR / Cas system may be non-naturally occurring. The CRISPR / Cas system may be engineered. The CRISPR / Cas system may be for modifying a genome at a cold temperature. The CRISPR / Cas system may comprise a cold-tolerant Cas effector or functional fragment thereof. The CRISPR / Cas system may comprise a guide RNA. The guide RNA may target the Cas effector to a target site in the genome.

[0072] In one aspect, provided herein is a CRISPR / Cas system for modifying a genome at a cold temperature, comprising a cold-tolerant Cas effector or functional fragment thereof and a guide RNA, wherein the guide RNA targets the Cas effector to a target site in the genome.Modifying a genome

[0073] As used herein, the term “modifying a genome” encompasses genetic and epigenetic modifications. Epigenetic modifications can include, for example, histone modifications and / or modifications in DNA methylation.

[0074] In some embodiments, modifying a genome can result in a change in an expression level of a gene.

[0075] A gene can comprise one or more regulatory elements. Regulatory elements include, but are not limited to, promoters (including core promoter, proximal promoter, and distal promoter), enhancers (including proximal and distal enhancers), silencers, or terminators. Regulatory elements may be upstream or downstream of the coding sequence of the gene. Regulatory elements may contain various sequence motifs that interact with transcriptional machinery or distal enhancers or silencers in trans.

[0076] In some embodiments, the target site comprises a gene in the genome. In some embodiments, the target site comprises a regulatory element of a gene.Cold temperature

[0077] As used herein, the term “cold temperature” is with reference to normal environmental conditions.

[0078] In some embodiments, the cold temperature is below about 20QC. In some embodiments, the cold temperature is below about 15QC. In some embodiments, the cold temperature is below about 10QC. In some embodiments, the cold temperature is below about 5QC.

[0079] In some embodiments, the cold temperature is a sub-zero temperature. In some embodiments, the cold temperature is below about 0QC. In some embodiments, the cold temperature is below about -5QC.

[0080] In some embodiments, the cold temperature is between about -10QC and about 20QC. In some embodiments, the cold temperature is between about -10QC and about 15QC. In some embodiments, the cold temperature is between about -10QC and about 10QC. In some embodiments, the cold temperature is between about -10QC and about 5QC. In some embodiments, the cold temperature is between about -10QC and about 0QC. In some embodiments, the cold temperature is between about -10QC and about -5QC.

[0081] In some embodiments, the cold temperature is between about -5QC and about 20QC. In some embodiments, the cold temperature is between about -5QC and about 15QC. In some embodiments, the cold temperature is between about -5QC andabout 10QC. In some embodiments, the cold temperature is between about -5QC and about 5QC. In some embodiments, the cold temperature is between about -5QC and about 0QC.

[0082] In some embodiments, the cold temperature is between about 0QC and about 20QC. In some embodiments, the cold temperature is between about 0QC and about 15QC. In some embodiments, the cold temperature is between about 0QC and about 10QC. In some embodiments, the cold temperature is between about 0QC and about 5QC.

[0083] In some embodiments, the cold temperature is between about 5QC and about 20QC. In some embodiments, the cold temperature is between about 5QC and about 15QC. In some embodiments, the cold temperature is between about 5QC and about 10QC.

[0084] In some embodiments, the cold temperature is between about 10QC and about 20QC. In some embodiments, the cold temperature is between about 10QC and about 15QC.

[0085] In some embodiments, the cold temperature is between about 15QC and about 20QC.

[0086] In some embodiments, the cold temperature is about -10QC. In some embodiments, the cold temperature is about -9QC. In some embodiments, the cold temperature is about -8QC. In some embodiments, the cold temperature is about -7QC. In some embodiments, the cold temperature is about -6QC. In some embodiments, the cold temperature is about -5QC. In some embodiments, the cold temperature is about -4QC. In some embodiments, the cold temperature is about -3QC. In some embodiments, the cold temperature is about -2QC. In some embodiments, the cold temperature is about -1QC. In some embodiments, the cold temperature is about 0QC. In some embodiments, the cold temperature is about 1QC. In some embodiments, the cold temperature is about2QC. In some embodiments, the cold temperature is about 3QC. In some embodiments, the cold temperature is about 4QC. In some embodiments, the cold temperature is about5QC. In some embodiments, the cold temperature is about 6QC. In some embodiments, the cold temperature is about 7QC. In some embodiments, the cold temperature is about8QC. In some embodiments, the cold temperature is about 9QC. In some embodiments,the cold temperature is about 10QC. In some embodiments, the cold temperature is about 1 1QC. In some embodiments, the cold temperature is about 12QC. In some embodiments, the cold temperature is about 13QC. In some embodiments, the cold temperature is about 14QC. In some embodiments, the cold temperature is about 15QC. In some embodiments, the cold temperature is about 16QC. In some embodiments, the cold temperature is about 17QC. In some embodiments, the cold temperature is about 18QC. In some embodiments, the cold temperature is about 19QC. In some embodiments, the cold temperature is about 20 °-C.Cold-tolerant

[0087] As used herein, the term “cold-tolerant”, when referring to a Cas effector, means the Cas effector is capable of performing a desired function at a cold temperature.

[0088] For example, a cold-tolerant Cas effector can be a Cas effector that can perform a desired activity efficiently, such as nuclease activity, at or below about 15QC. A cold-tolerant Cas effector can be a Cas effector that can perform a desired activity at sub-zero temperatures. A cold-tolerant Cas effector may be a Cas effector that has optimal activity at cold temperature, or a Cas effector that has optimal activity outside of the cold temperature but nonetheless has sufficient activity at the cold temperature.

[0089] The cold-tolerant Cas effector may be derived from naturally occurring Cas orthologs from prokaryotes. For example, the cold-tolerant Cas effector may be derived from a psychrophile. Psychrophiles refer to organisms, such as bacteria or archaea, that inhabit cold environments. Such organisms can have optimal growth at cold temperatures, such as below around 15 °C. or they can be cold-tolerant. For example, a cold-tolerant psychrophile can be one that can survive below 0°C but can grow optimally at 20-25°C.

[0090] In some embodiments, the cold-tolerant Cas effector is derived from a psychrophile. In some embodiments, the psychrophile comprises a Psychroflexus species. In some embodiments, the Psychroflexus species comprises Psychroflexus torquis. In some embodiments, the cold-tolerant Cas effector is derived from Psychroflexus torquis.

[0091] The cold-tolerant Cas effector may be derived from a non-psychrophile (e.g. a mesophile). The non-psychrophile may, for example, grow optimally above 15 °C, but can survive at lower temperatures, such as 10 °C or less.

[0092] In some embodiments, the cold-tolerant Cas effector is derived from Vibrio natriegens.Cas effector

[0093] As used herein, the term “Cas effector” refers to a Cas protein that binds a target nucleic acid site specified by an RNA guide. The term encompasses variants and derivatives, such as catalytically-deficient or catalytically-dead variants (e.g. nuclease- deficient), catalytically modified variants (e.g. single-strand cleavage vs double-strand cleavage), and variants fused to another domain (e.g. to impart a different function). A Cas effector may or may not possess an enzymatic activity. The term encompasses proteins comprising natural amino acids, or analogues or derivatives thereof. The term encompasses modifications, such as modifications to the backbone and modifications to side chains.

[0094] The CRISPR / Cas system of the present disclosure may comprise a cold- tolerant Cas effector or a functional fragment thereof. As used herein, the term “functional fragment” means any fragment having a desired function. For example, a functional fragment of a cold-tolerant Cas9 can be any fragment of the Cas9 that displays DNA cleavage activity at cold temperature.

[0095] The Cas effector may be a class I or class II Cas protein. Class I CRISPR systems typically utilize multi-subunit effector complexes for target recognition, while class II systems typically rely on single-protein effect complexes. Within each class, there are different types and subtypes of Cas proteins. The Cas effector may be any type or subtype of Cas protein. For example, the Cas effector may be Cas9, Cas12a (formerly known as Cpf 1 ), or Cas13a.

[0096] In some embodiments, the Cas effector comprises a class I Cas protein. In some embodiments, the Cas effector comprises a class II Cas protein. In some embodiments, the Cas effector comprises a type II Cas protein. In some embodiments, the Cas effector comprises a type V Cas protein. In some embodiments, the Cas effectorcomprises a type VI Cas protein. In some embodiments, the Cas effector comprises Cas9. In some embodiments, the Cas effector comprises Cas9. In some embodiments, the Cas effector comprises a Cas12. In some embodiments, the Cas effector comprises Cas12a. In some embodiments, the Cas effector comprises a Cas13. In some embodiments, the Cas effector comprises Cas13a.

[0097] In some embodiments, the Cas effector is nuclease-deficient. In some embodiments, the Cas effector is nuclease-dead.

[0098] The Cas effector may modulate gene expression by different mechanisms, including but not limited to, gene modification, RNA modification (e.g. RNA editing by Cas 13), and transcriptional modulation.

[0099] Transcriptional modulation may comprise transcriptional activation and / or transcriptional repression. For example, a psychrophile-derived Cas9 may be engineered to abolish its nuclease activity. The nuclease-dead variant may be used with tripartite transcriptional activation effector, comprising VP64 derived from herpes simplex virus, p65 from human NF-KB, and Rta from Epstein-Barr virus (dCas9-VPR). dCas9-VPR can induce transcriptional activation of the target gene. The nuclease-dead variant may be fused with the p300 core domain of human histone acetyltransferase (dCas9-P300). dCas9-P300 may enhance gene expression by histone modification. To repress transcription, dCas9 may be fused to a transcriptional inhibitor (such as a Kruppel- associated box (KRAB) repressor domain) and / or a DNA methyltransferase (such as DNMT3A / B). dCas9 may also physically block transcription.

[0100] The psychrophile-derived Cas effector may also be engineered to comprise a domain that imparts a different function. For example, a nuclease-dead Cas9 variant may be fused to a deaminase domain. Such a derivative may possess base-editing activities.

[0101] In some embodiments, the Cas effector comprises a base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the base editor is a cytosine base editor. In some embodiments, the base editor is a synchronous programmable adenine and cytosine editor.

[0102] The psychrophile-derived Cas effector may be engineered to have one or more of nuclease, nickase, transposase, base editing, or prime editing activity.

[0103] In some embodiments, the Cas effector comprises Cas9.

[0104] In some embodiments, the Cas effector comprises Cas9 from Psychroflexus torquis (PtoCas9). For example, Cas9 from P. torquis can comprise an amino acid sequence of UniProt K4I9M9.

[0105] In some embodiments, the Cas effector comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 . In some embodiments, the Cas effector comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 . In some embodiments, the Cas effector comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 . In some embodiments, the Cas effector comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 . In some embodiments, the Cas effector comprises an amino acid sequence of SEQ ID NO: 1.

[0106] In some embodiments, the Cas effector comprises Cas9 from Vibrio natriegens (VnaCas9).

[0107] In some embodiments, the Cas effector comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the Cas effector comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6. In some embodiments, the Cas effector comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the Cas effector comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the Cas effector comprises an amino acid sequence of SEQ ID NO: 6.Guide RNA

[0108] As used herein, the term “guide RNA” refers to any RNA molecule that facilities targeting of a Cas effector to a target nucleic acid.

[0109] A guide RNA may comprise a direct repeat sequence capable of forming a complex with a Cas effector and a spacer sequence that mediates target recognition, for example, by hybridizing to the target nucleic acid.

[0110] In some embodiments, the guide RNA targets PtoCas9 to the target nucleic acid.

[0111] In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 80% sequence identity to SEQ ID NO: 25. In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 85% sequence identity to SEQ ID NO: 25. In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 90% sequence identity to SEQ ID NO: 25. In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 95% sequence identity to SEQ ID NO: 25. In some embodiments, the guide RNA comprises a direct repeat sequence that has the sequence of SEQ ID NO: 25. In some embodiments, the guide RNA targets PtoCas9 to the target nucleic acid.

[0112] In some embodiments, the guide RNA targets VnaCas9 to the target nucleic acid.

[0113] In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 80% sequence identity to SEQ ID NO: 26. In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 85% sequence identity to SEQ ID NO: 26. In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 90% sequence identity to SEQ ID NO: 26. In some embodiments, the guide RNA comprises a direct repeat sequence that has at least about 95% sequence identity to SEQ ID NO: 26. In some embodiments, the guide RNA comprises a direct repeat sequence that has the sequence of SEQ ID NO: 26.

[0114] The CRISPR / Cas system of the present disclosure may comprise more than one guide RNA. The more than one guide RNA may target the same gene. The more than one guide RNA my target different genes. The more than one guide RNA may be provided as a single RNA molecule (referred to as “single guide RNA” or “sgRNA”), or as separate RNA molecules.Expression cassettes and vectors

[0115] In another aspect, provided herein are expression cassettes comprising a nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system disclosed herein, operably linked to a promoter.

[0116] “Operably linked” means the nucleic acid is linked to the promoter, and / or other regulatory elements, in such a manner that the nucleic acid is capable of being expressed.

[0117] The nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof may be codon-optimized for expression in a host cell.

[0118] In some embodiments, the cold-tolerant Cas effector is PtoCas9.

[0119] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 2.

[0120] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 3.

[0121] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 4.

[0122] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 5.

[0123] In some embodiments, the cold-tolerant Cas effector is VnaCas9.

[0124] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 7.

[0125] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80%sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 8.

[0126] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 9.

[0127] In some embodiments, the nucleic acid encoding the cold-tolerant Cas effector comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 10.

[0128] In another aspect, provided herein are expression cassettes comprising a nucleic acid encoding the guide RNA of the CRISPR / Cas system disclosed herein, operably linked to a promoter.

[0129] In some embodiments, the guide RNA is capable of targeting PtoCas9 to a target nucleic acid.

[0130] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 11 .

[0131] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 12.

[0132] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 13.

[0133] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 14.

[0134] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 15.

[0135] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 16.

[0136] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 17.

[0137] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 18.

[0138] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 19.

[0139] In some embodiments, the guide RNA is capable of targeting VnaCas9 to a target nucleic acid.

[0140] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 20.

[0141] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 21 .

[0142] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 22.

[0143] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 23.

[0144] In some embodiments, the nucleic acid encoding the guide RNA comprises a sequence with at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity with SEQ ID NO: 24.

[0145] In another aspect, provided herein are vectors comprising one or more expression cassettes disclosed herein.

[0146] The vector may be a viral vector. Viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus (AAV) vectors, lentiviral vectors, and retroviral vectors. The vector may be a non-viral vector, such as a plasmid.II. Methods

[0147] Disclosed herein, are methods of modifying a genome of a cell at a cold temperature. The method may comprise contacting the cell with a CRISPR / Cas system. The CRISPR / Cas system may be non-naturally occurring. The CRISPR / Cas system may be engineered. The CRISPR / Cas system may comprise a cold-tolerant Cas effector or functional fragment thereof. The CRISPR / Cas system may comprise a guide RNA. The guide RNA may target the Cas effector to a target site in the genome, thereby modifying the genome at the cold temperature.

[0148] In one aspect, provided herein are methods of modifying a genome of a cell at a cold temperature, comprising contacting the cell with a CRISPR / Cas system comprising a cold-tolerant Cas effector or functional fragment thereof, and a guide RNA,wherein the guide RNA targets the Cas effector or functional fragment thereof to a target site in the genome, thereby modifying the genome at the cold temperature.

[0149] Modifying a genome may result in a change in an expression level of a gene in the genome. Accordingly, the methods disclosed herein may be used to modulation expression of a gene in a cell.Contacting the cell with a CRISPR / Cas system

[0150] Contacting the cell with a CRISPR / Cas system may comprise delivering the CRISPR / Cas system by any suitable vehicle. For example, the Cas effector or functional fragment thereof and / or the guide RNA may be delivered using a vector comprising an expression cassette for expression of the Cas effector or functional fragment thereof and / or guide RNA. mRNA-based delivery may be used. The mRNA may be modified with, for example, 5’-cap, poly(A) tail, modified bases, modified ribose, and any combination thereof. The Cas effector protein of functional fragment thereof and the guide RNA may be complexed into a ribonucleoprotein for delivery.

[0151] The term "expression cassette", as used herein, refers to a DNA molecule comprising a nucleotide sequence that is expressed (transcribed) upon transfer into a cell, tissue or organ. Typically, an expression cassette comprises at least one gene (which may include a promoter, an open reading frame, and a termination signal). In some embodiments provided herein, the expression cassettes comprise, for example, polynucleotides that encode guide RNA(s) and / or Cas effector(s) or derivatives thereof, and optionally, cDNA(s) encoding gene(s)-of-interest as well as upstream promoter and downstream termination signal.

[0152] Various promoters are suitable for the expression cassettes provided herein. For example, Cas genes may be expressed from a CMV promoter, and guide RNAs may be expressed from a U6 promoter or an H1 promoter.

[0153] Expression cassettes comprising polynucleotides encoding Cas effector(s), guide RNA(s) and / or cDNA of a gene or genes of interest may be delivered to a cell, tissue or organ using any suitable vector, as known to those of ordinary skill in the art. The vector may be a viral vector. The vector may be a cloning vector. The vector may be a plasmid, phagemid, cosmid, or the like. In some embodiments, the vector is a viral vector. Viralvectors include, but are not limited to, an adenoviral vector, an adeno- associated virus (AAV) vector, a lentiviral vector, or a retroviral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the vector is an adenoviral vector.

[0154] In some embodiments, the vector includes a nucleic acid encoding a single component of the CRISPR / Cas system disclosed herein. In some embodiments, the vector comprises a plurality of nucleic acids, each encoding a different component of the CRISPR / Cas system disclosed herein.Cell

[0155] The methods disclosed herein may be used to modify a genome and / or modulate expression of a gene in a cell in vitro, in vivo or ex vivo. The cell may be a prokaryotic cell. The cell may be a eukaryotic cell, such as a human cell, a non-human animal cell (e.g. an insect cell), or a plant cell.

[0156] The cell may be a cell of a tissue or an organ. The tissue or organ may be a tissue or an organ to be used for transplantation. For example, organs may include but are not limited to lung, heart, kidney, liver, pancreas, stomach and intestine. Tissues may include, but are not limited to, cornea, bone, tendon, skin, pancreas islets, heart valves, nerves and veins.

[0157] In some embodiments, the tissue or organ is the lung.

[0158] The tissue or organ may be from human or any animal source, including but not limited to pig. The tissue or organ may be engineered, for example, re-cellularized scaffold.

[0159] The tissue or organ may be at a hypothermic state. Hypothermic state refers to conditions where a cell, a tissue or an organ is kept at below physiological temperatures. Keeping a cell, a tissue or an organ at a hypothermic state may preserve the cell, the tissue or the organ, for example, prior to transplantation.

[0160] A hypothermic state may comprise a temperature below physiological temperature.

[0161] In some embodiments, the hypothermic state comprises a temperature of below about 20QC. In some embodiments, the hypothermic state comprises a temperature of below about 15QC. In some embodiments, the hypothermic statecomprises a temperature of below about 10QC. In some embodiments, the hypothermic state comprises a temperature of below about 5QC. In some embodiments, the hypothermic state comprises a temperature of below about 0QC. In some embodiments, the hypothermic state comprises a temperature of below about -5QC.

[0162] In some embodiments, the hypothermic state comprises a temperature of between about -10QC and about 20QC. In some embodiments, the hypothermic state comprises a temperature of between about -10QC and about 15QC. In some embodiments, the hypothermic state comprises a temperature of between about -10QC and about 10QC. In some embodiments, the hypothermic state comprises a temperature of between about -10QC and about 5QC. In some embodiments, the hypothermic state comprises a temperature of between about -10QC and about 0QC. In some embodiments, the hypothermic state comprises a temperature of between about -10QC and about -5QC.

[0163] In some embodiments, the hypothermic state comprises a temperature of between about -5QC and about 20QC. In some embodiments, the hypothermic state comprises a temperature of between about -5QC and about 15QC. In some embodiments, the hypothermic state comprises a temperature of between about -5QC and about 10QC. In some embodiments, the hypothermic state comprises a temperature of between about -5QC and about 5QC. In some embodiments, the hypothermic state comprises a temperature of between about -5QC and about 0QC.

[0164] In some embodiments, the hypothermic state comprises a temperature of between about 0QC and about 20QC. In some embodiments, the hypothermic state comprises a temperature of between about 0QC and about 15QC. In some embodiments, the hypothermic state comprises a temperature of between about 0QC and about 10QC. In some embodiments, the hypothermic state comprises a temperature of between about 0QC and about 5QC.

[0165] In some embodiments, the hypothermic state comprises a temperature of between about 5QC and about 20QC. In some embodiments, the hypothermic state comprises a temperature of between about 5QC and about 15QC. In some embodiments, the hypothermic state comprises a temperature of between about 5QC and about 10QC.

[0166] In some embodiments, the hypothermic state comprises a temperature of between about 10QC and about 20QC. In some embodiments, the hypothermic state comprises a temperature of between about 10QC and about 15QC.

[0167] In some embodiments, the hypothermic state comprises a temperature of between about 15QC and about 20QC.

[0168] In some embodiments, the hypothermic state comprises a temperature of about -10QC. In some embodiments, the hypothermic state comprises a temperature of about -9QC.some embodiments, the hypothermic state comprises a temperature of about -8QC.some embodiments, the hypothermic state comprises a temperature of about -7QC.some embodiments, the hypothermic state comprises a temperature of about -6QC.some embodiments, the hypothermic state comprises a temperature of about -5QC.some embodiments, the hypothermic state comprises a temperature of about -4QC.some embodiments, the hypothermic state comprises a temperature of about -3QC.some embodiments, the hypothermic state comprises a temperature of about -2QC.some embodiments, the hypothermic state comprises a temperature of about -1QC.some embodiments, the hypothermic state comprises a temperature of about 0QC.some embodiments, the hypothermic state comprises a temperature about 1QC. In some embodiments, the hypothermic state comprises a temperature of about 2QC. In some embodiments, the hypothermic state comprises a temperature of about 3QC. In some embodiments, the hypothermic state comprises a temperature of about 4QC. In some embodiments, the hypothermic state comprises a temperature about 5QC. In some embodiments, the hypothermic state comprises a temperature of about 6QC. In some embodiments, the hypothermic state comprises a temperature of about 7QC. In some embodiments, the hypothermic state comprises a temperature of about 8QC. In some embodiments, the hypothermic state comprises a temperature of about 9QC. In some embodiments, the hypothermic state comprises a temperature about 10QC. In some embodiments, the hypothermic state comprises a temperature of aboutQC. In some embodiments, the hypothermic state comprises a temperature of about 12QC. In some embodiments, the hypothermic state comprises a temperature of about 13QC. In some embodiments, the hypothermic state comprises a temperature of about 14QC. In some embodiments, the hypothermic state comprises a temperature of about 15QC. In some embodiments, the hypothermic state comprises a temperature ofabout 16QC. In some embodiments, the hypothermic state comprises a temperature of about 17QC. In some embodiments, the hypothermic state comprises a temperature of about 18QC. In some embodiments, the hypothermic state comprises a temperature of about 19QC. In some embodiments, the hypothermic state comprises a temperature of about 20QC.Delivering to a tissue or organ via ex vivo organ perfusion (EVOP)

[0169] The methods disclosed herein may be used to modify the genome of a cell in a tissue or organ. The method may comprise delivering the CRISPR / Cas system via ex vivo organ perfusion (EVOP).

[0170] In some embodiments, contacting the cell with the CRISPR / Cas system comprises performing ex vivo organ perfusion (EVOP).

[0171] For example, an EVOP system can be used to deliver a vector comprising an expression cassette for expression of the Cas effector and / or guide RNA to the tissue or organ. The vector may be included in a perfusate.

[0172] Any perfusion system may be used. For example, certain systems are described in U.S. Patent Application No. 14 / 658,456, which discloses an ex vivo perfusion system and is incorporated herein by reference in its entirety. See also XVIVO PERFUSION, http: / / www.xvivoperfusion.com.

[0173] Where EVOP is performed on a tissue or organ for transplantation, EVOP may be used at any stage of the process to assist in the preservation, maintenance, repair, improvement, and / or assessment of the tissue or organ. For example, EVOP can be used soon after donor organ has been excised from the donor and can replace or supplement standard evaluation of process. EVOP can also be used after standard evaluation on both transplantable organ and injured organ in order to preserve, maintain, improve, assess and, in the case of injured organ, repair donor organs. Tissues and organs subjected to EVOP may be preserved and maintained for 12, 24, 72 hours, or longer.

[0174] In some embodiments, the EVOP is ex vivo lung perfusion (EVLP).

[0175] Where delivery of the CRISPR / Cas system disclosed herein comprises delivery of a vector (for example, adenoviral vectors comprising expression cassettes that encode one or more guide RNA(s) and a Cas effector) to a lung, a bronchoscope, suchas a flexible fibre-optic bronchoscope, can be inserted through an endotracheal tube inserted in the lungs during EVLP. A fine catheter can then be inserted through the bronchoscope channel and can be used to deliver the vector(s) into each segmental bronchus.Cold static preservation of a tissue or an organ

[0176] Prior to EVOP, a tissue or an organ may be subject to cold static preservation. EVOP may be performed at normothermic conditions, for example, 37QC. After EVOP, the tissue or organ may again be subject to cold static preservation.

[0177] In some embodiments, the method further comprises preserving the tissue or organ at a first temperature before performing the EVOP.

[0178] In some embodiments, the first temperature comprises a temperature of below about 20QC. In some embodiments, the first temperature comprises a temperature of below about 15QC. In some embodiments, the first temperature comprises a temperature of below about 10QC. In some embodiments, the first temperature comprises a temperature of below about 5QC. In some embodiments, the first temperature comprises a temperature of below about 0QC. In some embodiments, the first temperature comprises a temperature of below about -5QC.

[0179] In some embodiments, the first temperature comprises a temperature of about -10QC. In some embodiments, the first temperature comprises a temperature of about -9QC. In some embodiments, the first temperature comprises a temperature of about -8QC. In some embodiments, the first temperature comprises a temperature of about -7QC. In some embodiments, the first temperature comprises a temperature of about -6QC. In some embodiments, the first temperature comprises a temperature of about -5QC. In some embodiments, the first temperature comprises a temperature of about -4QC. In some embodiments, the first temperature comprises a temperature of about -3QC. In some embodiments, the first temperature comprises a temperature of about -2QC. In some embodiments, the first temperature comprises a temperature of about -1QC. In some embodiments, the first temperature comprises a temperature of about 0QC. In some embodiments, the first temperature comprises a temperature of about 1QC. In some embodiments, the first temperature comprises a temperature of about 2QC. In some embodiments, the first temperature comprises a temperature of about 3QC. Insome embodiments, the first temperature comprises a temperature of about 4QC. In some embodiments, the first temperature comprises a temperature of about 5QC. In some embodiments, the first temperature comprises a temperature of about 6QC. In some embodiments, the first temperature comprises a temperature of about 7QC. In some embodiments, the first temperature comprises a temperature of about 8QC. In some embodiments, the first temperature comprises a temperature of about 9QC. In some embodiments, the first temperature comprises a temperature of about 10QC. In some embodiments, the first temperature comprises a temperature of about 11QC. In some embodiments, the first temperature comprises a temperature of about 12QC. In some embodiments, the first temperature comprises a temperature of about 13QC. In some embodiments, the first temperature comprises a temperature of about 14QC. In some embodiments, the first temperature comprises a temperature of about 15QC. In some embodiments, the first temperature comprises a temperature of about 16QC. In some embodiments, the first temperature comprises a temperature of about 17QC. In some embodiments, the first temperature comprises a temperature of about 18QC. In some embodiments, the first temperature comprises a temperature of about 19QC.

[0180] In some embodiments, the method further comprises preserving the tissue or organ at a second temperature after performing the EVOP.

[0181] In some embodiments, the second temperature comprises a temperature of below about 20QC. In some embodiments, the second temperature comprises a temperature of below about 15QC. In some embodiments, the second temperature comprises a temperature of below about 10QC. In some embodiments, the second temperature comprises a temperature of below about 5QC. In some embodiments, the second temperature comprises a temperature of below about 0QC. In some embodiments, the second temperature comprises a temperature of below about -5QC.

[0182] In some embodiments, the second temperature comprises a temperature of about -10QC. In some embodiments, the second temperature comprises a temperature of about -9QC. In some embodiments, the second temperature comprises a temperature of about -8QC. In some embodiments, the second temperature comprises a temperature of about -7QC. In some embodiments, the second temperature comprises a temperature of about -6QC. In some embodiments, the second temperature comprises a temperatureof about -5QC. In some embodiments, the second temperature comprises a temperature of about -4QC. In some embodiments, the second temperature comprises a temperature of about -3QC. In some embodiments, the second temperature comprises a temperature of about -2QC. In some embodiments, the second temperature comprises a temperature of about -1QC. In some embodiments, the second temperature comprises a temperature of about 0QC. In some embodiments, the second temperature comprises a temperature of about 1QC. In some embodiments, the second temperature comprises a temperature of about 2QC. In some embodiments, the second temperature comprises a temperature of about 3QC. In some embodiments, the second temperature comprises a temperature of about 4QC. In some embodiments, the second temperature comprises a temperature of about 5QC. In some embodiments, the second temperature comprises a temperature of about 6QC. In some embodiments, the second temperature comprises a temperature of about 7QC. In some embodiments, the second temperature comprises a temperature of about 8QC. In some embodiments, the second temperature comprises a temperature of about 9QC. In some embodiments, the second temperature comprises a temperature of about 10QC. In some embodiments, the second temperature comprises a temperature of about 11QC. In some embodiments, the second temperature comprises a temperature of about 12QC. In some embodiments, the second temperature comprises a temperature of about 13QC. In some embodiments, the second temperature comprises a temperature of about 14QC. In some embodiments, the second temperature comprises a temperature of about 15QC. In some embodiments, the second temperature comprises a temperature of about 16QC. In some embodiments, the second temperature comprises a temperature of about 17QC. In some embodiments, the second temperature comprises a temperature of about 18QC. In some embodiments, the second temperature comprises a temperature of about 19QC.Target site

[0183] The target site may be within a gene or outside of a gene. The target site may be a coding region or a non-coding region.

[0184] A gene can comprise one or more regulatory elements. Regulatory elements include, but are not limited to, promoters (including core promoter, proximal promoter, and distal promoter), enhancers (including proximal and distal enhancers),silencers, or terminators. Regulatory elements may be upstream or downstream of the coding sequence of the gene. Regulatory elements may contain various sequence motifs that interact with transcriptional machinery or distal enhancers or silencers in trans.

[0185] In some embodiments, the target site comprises a gene in the genome. In some embodiments, the target site comprises a regulatory element of a gene.

[0186] In some embodiments, modifying a genome can result in a change in an expression level of a gene.

[0187] The gene whose expression can be modulated using the method disclosed herein can be any gene in the cell. For example, the gene may be a gene involved in the immune and / or inflammatory response. The gene may be a gene encoding a cytokine. The gene may include any inflammation-regulating and / or immune regulating genes. For example, the inflammation-regulating and / or immune regulating genes may include, but are not limited to: interleukin-10 (IL-10), interleukin 1 receptor antagonist (IL-1 RN), lnterleukin-4 (IL-4), lnterleukin-6 (IL-6), lnterleukin-8 (IL-8), tumor necrosis factor binding protein (TNFBP), cytotoxic T-lymphocyte associated protein 4 (CTLA4), NFKB inhibitor alpha (Ikba), Programmed cell death-1 (PD-1 ), Programmed cell death-ligand 1 (PD-L1 ), Interleukin-10 receptor, CD47, CD200, Fas Ligand (FasL), MHC molecule (MHC-lb) H2- M3, Serine protease inhibitor 6 (Spi6), C-C Motif Chemokine Ligand 21 (Ccl21 ), Milk Fat Globule-EGF Factor 8 Protein (Mfge8).

[0188] In some embodiments, the target site comprises a gene involved in the immune and / or inflammatory response. In some embodiments, the target site comprises a gene encoding a cytokine. In some embodiments, the target site comprises a gene encoding IL-10.III. Definitions

[0189] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0190] The term “or” is used to mean “and / or”, unless it is indicated explicitly to refer to alternatives only. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the expression “A, B, and / or C” can mean A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.

[0191] The term “comprise”, “comprising”, or the like means additional elements or components other than those recited may be present. Other terms such as “include”, “contain”, “have” and the like have similar meaning.

[0192] The term “consist of”, “consisting of” or the like means no additional component is present.

[0193] As used herein, the singular forms “a,” “an,” and “the” include plural references, unless indicated otherwise. For example, a reference to “a molecule” can be a reference to more than one molecule.

[0194] Further, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0195] More specifically, the term “about” means plus or minus 0.1 to 20%, 5-20%, or 10-20%, 10%-15%, preferably 5-10%, most preferably about 5% of the number to which reference is being made. The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1 .5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0196] The term “sequence identity”, in the context of nucleotide or amino acid sequences, refers to the percentage of nucleotides or amino acid residues that are identical between two sequences when aligned. Alignment may be in such a way to provide the highest level of matches between the two sequences. The comparison may be over the entire length of a sequence, or over a specific portion of a sequence. Gappedalignments may be permitted. Sequence identity may be determined manually, or by a sequence comparison algorithm. When determined manually, after aligning the two sequences, the number of identical positions can be divided by the number of nucleotides or amino acid residues in the reference sequence. Therefore, for example, a sequence identity of at least 90% means a sequence is identical to the reference sequence at at least 90 out of 100 positions of the reference sequence. Various computer programs and online tools may be used to determine sequence identity and are known in the art. For example, the basic local alignment search tool (“BLAST”; Altschul et al., (1997) Nucleic Acids Res., 25: 3389-3402) can be available through the National Center for Biotechnology Information (“NCBI”). When using computer programs or algorithms, default parameters are generally used.

[0197] The term “operably linked” means that two components are coupled in a manner so that one component affects the function of the other component. For example, a sequence can be operably linked to a regulatory element such that the regulatory element can regulate the expression of the sequence.

[0198] The term “regulatory element” refers to a sequence capable to controlling expression of a nucleic acid and can include controlling at the level of transcription and translation. Examples of regulatory elements can include, without limitation, promoters, enhancers, terminators, poly-A signals, 5’-UTR, 3’-UTR, IRES, etc.

[0199] The term “nucleic acid” or “nucleic acid molecule” as used herein means a polymer of at least two nucleotides. A nucleic acid may be single-stranded or doublestranded, or may contain portions of both. For example, a nucleic acid may have a stemloop structure, where the stem portion is double-stranded and the loop portion is singlestranded. A nucleic acid may be DNA, RNA, or a hybrid. A nucleic acid may be in different conformations, such as linear or circular.

[0200] The terms “polypeptide”, “peptide” and “protein” are used interchangeably and refer to a polymer of at least two amino acids. The amino acids may be natural amino acids, or analogues or derivatives thereof. The term encompasses modifications, such as modifications to the backbone and modifications to side chains.

[0201] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0202] Although any compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of compositions and methods are now described.EXAMPLESExample 1 - Impact of low temperature on DNA cleavage activity by SpyCas9

[0203] In vitro cleavage assay using purified SpyCas9 RNP was performed. DNA substrate and RNP were incubated at 37QC, 15QC, 10QC, or 4QC, and cleavage efficiency at different time points was determined. Figs. 1 A-1 C show that at low temperatures, the efficiency of SpyCas9 is significantly reduced.Example 2 - CRISPR / Cas system derived from Psychroflexus torquis

[0204] Genome analysis of P. torquis identified a Cas9 gene, which is 4530 bp in size, and CRISPR array, which contain 24 sets of spacer and repeat sequences (Fig. 2A). A putative tracrRNA was identified. A crRNA and tracrRNA duplex was computationally predicted (Fig. 2B). A sgRNA was designed (Fig. 2C). PAM was predicted (PAM1 : TAATAAT; PAM2: TTATAAT; PAM3: TAATTAT; PAM4: TTATTAT; see Fig. 2D).Example 3 - In vitro cleavage assays

[0205] Cold tolerance of the engineered Cas systems will be evaluated by examining DNA cleavage efficiency at 4°C, 10°C, 15°C, and 37°C compared with Streptococcus pyogenes Cas9 (SpyCas9), a widely used Cas ortholog from mesophiles, using purified ribonucleoprotein (RNP), a complex of Cas protein and sgRNA.Example 4 - Evaluate the genome-editing efficiency at low temperatures in human cells

[0206] Genome-editing efficiency of the engineered Cas systems in HEK293 cells will be evaluated using a low-temperature culture protocol. Editing enzymes will be expressed in cells by plasmid transfection. After 24 h, when the genome editing initiates, the treated cells will be transferred to 4°C or 10°C and incubated for 12 h, and thentransferred to 37°C again. The editing efficiency will be analyzed at different time points using next-generation sequencing and compared with SpyCas9.Example 5 - Using cold-tolerant CRISPR system to modify donor lungs during cold preservation

[0207] Cas gene, CRISPR RNA, and trans-activating RNA can be identified in the genome sequence of a psychrophile. Protospacer adjacent motif can be computationally predicted. The kinetics and efficiency of the identified editor can be evaluated by in vitro DNA cleavage analysis at various temperatures. Finally, the editing can be assessed in HEK cell line using a low-temperature culture model. The psychrophilic Cas enzyme can be used to genetically modify donor lungs during cold preservation to provide immediate and long-lasting graft immunomodulation in recipients (Figs. 3A-3B, Figs. 4A-4B).

[0208] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0209] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and / or biomarker sequences (e.g. protein and / or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

[0210] The scope of the claims should not be limited by the embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS:1 . A non-naturally occurring CRISPR / Cas system for modifying a genome of a cell at a cold temperature, comprising a cold-tolerant Cas effector or functional fragment thereof and a guide RNA, wherein the guide RNA targets the Cas effector to a target site in the genome.

2. The system of claim 1 , wherein the cold temperature comprises a temperature below about 20QC, below about 15QC, below about 10QC, below about 5QC, below about 0QC, or below about -5QC.

3. The system of claim 1 or 2, wherein the Cas effector comprises a Class II Cas protein.

4. The system of any one of claims 1 -3, wherein the Cas effector comprises a type II Cas protein, a type V Cas protein, or a type VI Cas protein.

5. The system of any one of claims 1 -4, wherein the Cas effector comprises Cas9, Cas12a, and / or Cas13a.

6. The system of any one of claims 1 -5, wherein the Cas effector comprises Cas9.

7. The system of any one of claims 1 -6, wherein the cold-tolerant Cas effector is derived from a psychrophile.

8. The system of claim 7, wherein the psychrophile comprises a Psychroflexus.

9. The system of claim 8, wherein the Psychroflexus comprises Psychroflexus torquis.

10. The system of any one of claims 1 -7, wherein the cold-tolerant Cas effector comprises Psychroflexus torquis Cas9 (PtoCas9).1 1 . The system of any one of claims 1 -7, wherein the cold-tolerant Cas effector comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 1 .

12. The system of any one of claims 1 -6, wherein the cold-tolerant Cas effector is derived from Vibrio natriegens.

13. The system of any one of claims 1 -6, wherein the cold-tolerant Cas effector comprises Vibrio natriegens Cas9 (VnaCas9).

14. The system of any one of claims 1 -6, wherein the cold-tolerant Cas effector comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 6.

15. The system of any one of claims 1 -6, wherein the guide RNA targets PtoCas9 to a target site in the genome.

16. The system of any one of claims 1 -6, wherein the guide RNA comprises a sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 25.

17. The system of any one of claims 1 -6, wherein guide RNA targets VnaCas9 to a target site in the genome.

18. The system of any one of claims 1 -6, wherein guide RNA comprises a sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 26.

19. The system of any one of claims 1 -18, wherein the cell is a prokaryotic cell.

20. The system of any one of claims 1 -18, wherein the cell is a eukaryotic cell.21 . The system of any one of claims 1 -18, wherein the cell is a human cell.

22. The system of any one of claims 1 -18, wherein the cell is a non-human animal cell.

23. An expression cassette comprising a nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the system of any one of claims 1 -22.

24. The expression cassette of claim 23, wherein the nucleic acid encoding the cold- tolerant Cas effector or functional fragment thereof is operably linked to a promoter.

25. The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 2.

26. The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 3.

27. The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 4.

28. The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 5.

29. The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 7.

30. The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 8.31 . The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 9.

32. The expression cassette of claim 22 or 23, wherein the nucleic acid encoding the cold-tolerant Cas effector or functional fragment thereof of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 10.

33. An expression cassette comprising a nucleic acid encoding the guide RNA of the CRISPR / Cas system of any one of claims 1 -22,34. The expression cassette of claim 33, wherein the nucleic acid encoding the guide RNA is operably linked to a promoter.

35. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 11 .

36. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 12.

37. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 13.

38. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 14.

39. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 15.

40. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 16.41 . The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 17.

42. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 18.

43. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 19.

44. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 20.

45. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 21 .

46. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 22.

47. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 23.

48. The expression cassette of claim 33 or 34, wherein the nucleic acid encoding the guide RNA of the CRISPR / Cas system comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 24.

49. A vector comprising the expression cassette of any one of claims 23-48.

50. The vector of claim 49, wherein the vector comprises a viral vector.51 . The vector of claim 49, wherein the vector comprises a non-viral vector.

52. A method of modifying a genome of a cell at a cold temperature, comprising contacting the cell with a CRISPR / Cas system comprising a cold-tolerant Cas effector or functional fragment thereof, and a guide RNA, wherein the guide RNA targets the Cas effector or functional fragment thereof to a target site in the genome, thereby modifying the genome at the cold temperature.

53. A method of modifying a genome of a cell at a cold temperature, comprising contacting the cell with the system of any one of claims 1 -22, thereby modifying the genome at the cold temperature.

54. The method of claim 52 or 53, wherein the cold temperature comprises a temperature below about 20QC, below about 15QC, below about 10QC, below about 5QC, below about 0QC, or below about -5QC.

55. The method of any one of claims 52-54, wherein the Cas effector comprises a Class II Cas protein.

56. The method of any one of claims 52-55, wherein the Cas effector comprises a type II Cas protein, a type V Cas protein, or a type VI Cas protein.

57. The method of any one of claims 52-56, wherein the Cas effector comprises Cas9, Cas12a, and / or Cas13a.

58. The method of any one of claims 52-57, wherein the Cas effector comprises Cas9.

59. The method of any one of claims 52-58, wherein the cold-tolerant Cas effector is derived from a psychrophile.

60. The method of claim 59, wherein the psychrophile comprises a Psychroflexus.61 . The method of claim 60, wherein the Psychroflexus comprises Psychroflexus torquis.

62. The method of any one of claims 52-61 , wherein the cold-tolerant Cas effector comprises Psychroflexus torquis Cas9 (PtoCas9).

63. The method of any one of claims 52-58, wherein the cold-tolerant Cas effector is derived from Vibrio natriegens.

64. The method of any one of claims 52-58, wherein the cold-tolerant Cas effector comprises Vibrio natriegens Cas9 (VnaCas9).

65. The method of any one of claims 52-64, wherein the cell is a prokaryotic cell.

66. The method of any one of claims 52-64, wherein the cell is a eukaryotic cell.

67. The method of any one of claims 52-64, wherein the cell is a human cell.

68. The method of any one of claims 52-64, wherein the cell is a non-human animal cell.

69. The method of any one of claims 52-64, wherein the cell is a cell of a tissue or an organ.

70. The method of claim 69, wherein the tissue or organ is a tissue or organ for transplantation.71 . The method of claim 70, wherein tissue or organ is at a hypothermic state.

72. The method of claim 71 , wherein hypothermic state comprises a temperature of between about 0QC and about 10QC, or between about 4QC and about 10QC.

73. The method of claim 71 , wherein hypothermic state comprises sub-zero temperatures.

74. The method of claim 73, wherein hypothermic state comprises a temperature of between about -10QC and about 10QC, between about -8QC and about 10QC, between about -6QC and about 10QC, between about -4QC and about 10QC or between about -2QC and about 10QC.

75. The method of any one of claims 69-74, wherein contacting the cell with the CRISPR / Cas system comprises performing ex vivo organ perfusion (EVOP).

76. The method of claim 75, wherein the method further comprises preserving the tissue or organ at a first temperature before performing the EVOP.

77. The method of claim 76, wherein the first temperature is about 10QC.

78. The method of claim 75 or 76, wherein the method further comprises preserving the tissue or organ at a second temperature before performing the EVOP.

79. The method of claim 78, wherein the second temperature is about 4QC or about 10eC.

80. The method of any one of claims 69-79, wherein the tissue or organ is the lung.81 . The method of any one of claims 75-80, wherein the EVOP is ex vivo lung perfusion (EVLP).

82. The method of any one of claims 52-81 , wherein modifying the genome of the cell results in a change in an expression level of a gene in the genome.

83. The method of any one of claims 52-82, wherein the target site comprises an inflammation-regulating and / or immune-regulating gene.

84. The method of any one of claims 52-82, wherein the target site comprises a gene encoding a cytokine.

85. The method of any one of claims 52-82, wherein the target site comprises a gene encoding IL-10.