Lipid nanoparticles and methods of use

Abstract

Lipid nanoparticle (LNP) formulations, methods of producing, and methods of use provided. In some cases, a subject LNP includes: (a) an ionizable lipid (e.g., BP lipid 312, LP01); (b) a neutral phospholipid (e.g., DOPE); (c) cholesterol; (d) a pegylated lipid (e.g., DMG-PEG-2000); and (e) a molecular payload. In some embodiments, a subject LNP includes: (a) a cationic lipid (e.g., ADC); (b) an ionizable lipid (e.g., Lipid III-45, ALC-0315); (c) a neutral phospholipid (e.g., DOPE); (d) cholesterol; (e) a pegylated lipid (e.g., DMG-PEG-2000); and (f) a molecular payload.

Description

LIPID NANOPARTICLES AND METHODS OF USECross-Reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 693,051 filed September 10, 2024, which application is incorporated herein by reference in its entirety.Statement Regarding Federally Sponsored Research

[0002] This invention was made with government support under Grant Number HG009490 awarded by the National Institutes of Health. The government has certain rights in the invention.Incorporation by reference of Sequence Listing provided as an XML File

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 540PRV_SEQ_LIST.xml” created on September 10, 2024 and having a size of 151 ,458 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.I. Introduction

[0004] CRISPR-Cas9-based genome editing has the potential to provide wide-ranging treatments for genetic diseases if safe and effective methods for delivering CRISPR- based therapeutics can be developed. Although viral delivery of CRISPR genome editors is the most widely used method for in vivo cell editing, viral vectors can be immunogenic, carry the risk of vector genome integration and can induce off-target DNA damage due to continuous genome editor expression. Alternative non-viral strategies for delivering CRISPR editors could address these limitations if issues of efficacy and toxicity can be overcome.

[0005] Lipid-nanoparticle (LNP):mRNA complexes are non-virally derived vehicles for in vivo delivery that have provided for genome editing in the liver. However, developing LNP:mRNA complexes that can edit non-liver tissues remains a challenge. Although LNPs can deliver mRNAs coding for Cre recombinase, luciferase, and fluorescent proteins to non-liver organs, making the transition from reporter enzymes to CRISPR mRNA and sgRNA delivery has been inefficient. LNP-mediated delivery of CRISPR mRNA and sgRNA faces challenges of sgRNA instability, mRNA-mediated Toll-like receptor (TLR) activation, and low translational efficiency of the large mRNAs encoding genome editors. These challenges are inherent to the mRNA formulationbut could possibly be mitigated with alternative LNP delivery cargoes and formulations.

[0006] This disclosure provides compositions and methods with increased efficiency for delivering a molecular payload such as CRISPR-Cas effector proteins, guide RNAs, and / nucleic acids encoding same.II. SUMMARY

[0007] Direct delivery of genome editors in the form of RNP complexes has the potential to address several of the limitations associated with mRNA and viral-based delivery of CRISPR editors. In particular, RNPs are expected to elicit lower levels of TLR activation than mRNA and produce minimal off-target DNA damage due to their short intracellular half-life. In addition, RNPs may offer higher in vivo editing efficiency compared to mRNA-based delivery methods by avoiding in-situ translation of large mRNA and providing natural protection of the sgRNA by high-affinity Cas9 binding.

[0008] Strategies for delivering RNPs include the use of complex polymers, silica nanoparticles, metal-organic frameworks, LNPs and other formulations. However, only LNPs have a proven track record of clinical use and established procedures for good manufacturing practice (GMP). A successful LNP-based delivery strategy for RNPs, therefore, has great translational potential.

[0009] The inventors realized that: (i) RNPs lack the negative charge density needed for efficient LNP encapsulation, (ii) conditions to formulate LNPs usually consist of organic solvents that can denature proteins, and (iii) although LNP-mediated delivery of SpyCas9 in the RNP format induced genome editing in the liver, delivery to nonliver organs such as the lungs remained inefficient.

[0010] The work described in the experimental examples below led to the surprising finding by the inventors of new lipid nanoparticle (LNP) formulations that provide for efficient delivery of a molecular payload (e.g., an mRNA encoding the CRISPR-Cas effector protein; a CRISPR-Cas effector protein, e.g., complexed with a guide RNA as a ribonucleoprotein (RNP); etc.) to cells and tissues, e.g., liver, lung, spleen, kidney, and / or heart cells / tissues.

[0011] In some cases, a CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to iGeoCas9(C) (SEQ ID NO: 34), which is the same as the wild type GeoCas9 of SEQ ID NO: 1 , but with the following mutations: E149G, T182I, N206D, P466Q, Q817R, E843K, E884G, andK908R (see, e.g., Chen et al., bioRxiv. Preprint. 2023 Nov 15: doi:10.1101 / 2023.11.15.566339). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to iGeoCas9(G)(SEQ ID NO: 35), which is the same as SEQ ID NO:1 , but with the following mutations: E149G, T182I, N206D, P466Q, E843K, E884G, K908R, T1015A, and D1017N (see, e.g., Chen et al., bioRxiv. Preprint. 2023 Nov 15: doi:10.1101 / 2023.11.15.566339). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to ThermoCas9 (R1W1)(SEQ ID NO: 36), which is the same as wild type ThermoCas9 (SEQ ID NO: 6), but includes iGeo-like mutations. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any of SEQ ID NOs.: 1-36.

[0012] Provided are lipid nanoparticles (LNPs) formulated for delivery of a payload to a target cell. In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes: (a) an ionizable lipid (e.g., BP lipid 312, LP01) present in a mole percentage of about 36-56% of the total lipids; (b) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 8-20% of the total lipids; (c) cholesterol present in a mole percentage of about 30-50% of the total lipids; (d) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 1-4% of the total lipids; and (e) a molecular payload. In some cases, the ionizable lipid is present in a mole percentage of about 46% of the total lipids. In some cases, the neutral phospholipid is present in a mole percentage of about 10-15% of the total lipids. In some cases, cholesterol is present in a mole percentage of about 40% of the total lipids. In some cases, the pegylated lipid is present in a mole percentage of about 1-2% of the total lipids. In some cases, the LNP further includes a pegylated cholesterol (e.g., Chol- PEG-2000). In some cases, the pegylated cholesterol is present in a mole percentage of about 0.2-1% (e.g., about 0.4%) of the total lipids. In some cases, a subject LNP does not include a cationic lipid.

[0013] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes: (a) a cationic lipid (e.g., ADC) present in a mole percentage of about 30- 55% of the total lipids; (b) an ionizable lipid (e.g., Lipid III-45, ALC-0315) present in a mole percentage of about 15-35% of the total lipids; (c) a neutral phospholipid (e.g.,DOPE) present in a mole percentage of about 8-20% of the total lipids; (d) cholesterol present in a mole percentage of about 10-25% of the total lipids; (e) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 0.8-4% of the total lipids; and (f) a molecular payload. In some cases, the cationic lipid is present in a mole percentage of about 40-48% (e.g., about 42%, about 46%) of the total lipids. In some cases, the ionizable lipid is present in a mole percentage of about 20-30% (e.g., about 24%, about 26%) of the total lipids. In some cases, the neutral phospholipid is present in a mole percentage of about 11-16% (e.g., about 12%, about 15%) of the total lipids. In some cases, cholesterol is present in a mole percentage of about 14- 20% (e.g., about 16%) of the total lipids. In some cases, the pegylated lipid is present in a mole percentage of about 1-3% (e.g., about 1 .5%) of the total lipids.

[0014] In some cases (e.g., for any of the LNP formulations discussed above), the molecular payload includes an mRNA encoding a CRISPR-Cas effector protein. In some cases, the molecular payload includes a CRISPR-Cas guide RNA and an mRNA encoding a CRISPR-Cas effector protein. In some cases, the molecular payload includes a CRISPR-Cas effector protein. In some cases, the molecular payload includes an RNP comprising a CRISPR-Cas effector protein complexed with a CRISPR-Cas guide RNA.

[0015] Also provided are methods of making and methods of delivering LNPs of the disclosure, e.g., delivery to liver, lung, spleen, kidney, and / or heart tissue of an individual. Reagents, compositions, and kits / systems that find use in practicing the subject methods are provided.III. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0017] FIG. 1A-1 F Directed evolution of GeoCas9 improves its editing efficiency by orders of magnitude and broadens its PAM compatibility, a. Schematic diagram of the direct evolution system used to evolve GeoCas9, based on bacterial selection, b. Evolutionary lineage of GeoCas9 mutants, c. Compared to the wild-type GeoCas9, evolved GeoCas9 (mutant R1 W1) well-preserved its thermostability with a melting temperature much higher than canonical SpyCas9. Melting temperatures of the three Cas9 proteins were measured via a thermal shift assay, d. Schematic diagram ofGeoCas9-mediated genome editing of NPCs isolated from Ai9 mice. The spacer and PAM sequences of the GeoCas9 gRNAs were designed to turn tdTomato on if successful editing occurs. Guides g7 and g8 target the LoxP sites for the stop cassette deletion, e. GeoCas9 mutants edit NPCs with significantly higher efficiency than wild-type GeoCas9 after electroporation-mediated delivery. Genome editing efficiencies quantified based on tdTom(+) signals with the whole lineage of GeoCas9 mutants paired with different sgRNAs. f. PAM specificity is broadened through the further engineering of the GeoCas9 PI domain, n = 4 for each group, data are presented as mean values with individual data points.

[0018] FIG. 2A-2B iGeoCas9 edits cells more efficiently than SpyCas9 or iCas12a after LNP- mediated delivery, a. iGeoCas9, SpyCas9, and iCas12a edit cells with similar efficiency after nucleofection. However, iGeoCas9 edits cells more efficiently after LNP-mediated delivery than either SpyCas9 or iCas12a. n = 4 for each group, data are presented as mean values with individual data points, b. Chemical structures of the lipids used in this study, two formulations were identified that delivered iGeoCas9 RNP efficiently, termed standard and cationic (see table for details). Dynamic light scattering (DLS) of standard and cationic LNPs demonstrate they have sizes of 178 nm and 181 nm. iGeoCas9 used in this figure is 2NLS-iGeoCas9(C2)-2NLS.

[0019] FIG. 3A-3D iGeoCas9 RNP:LNP complexes can edit a wide range of genomic targets and multiple different cell lines a. LNP-mediated delivery of iGeoCas9 RNPs edits neural progenitor cells (NPCs) with efficiencies comparable to nucleofection. b. Chemical modification of sgRNAs improves the editing efficiency after LNP-mediated delivery (ms = 2’-methoxy and phosphorothioate linkage), c. Comparison of the genome editing levels in HEK293T cells based on nucleofection and LNP-assisted delivery of iGeoCas9 RNP. (Left) Schematic diagram of iGeoCas9-mediated genome editing of HEK293T EGFP cells, resulting in the knock-out of EGFP fluorescence. (Right) Genome editing efficiencies quantified based on EGFP(-) signals using the engineered GeoCas9 paired with different sgRNAs. n = 4 for each group, data are presented as mean values with individual data points, d. iGeoCas9 RNP:LNP complexes exhibit ultra-stability, allowing for long-term storage in a neutral buffer at 4 °C. (Left) Schematic illustration of LNP stability test. (Right) Genome editing efficiencies quantified based on tdTom(+) or EGFP(-) signals using iGeoCas9 RNP:LNP complexes stored at 4 °C for certain amounts of time, n = 4 for each group, data are presented as mean values with standard deviations. iGeoCas9 used in this figure is 2NLS-iGeoCas9(C2)-2NLS.

[0020] FIG. 4A-4B Co-delivery of iGeoCas9 RNPs and ssDNA templates with LNPs efficiently generates HDR in cells, a. Characterization of LNPs encapsulating iGeoCas9 RNPs and ssDNA templates, b. Co-delivery of iGeoCas9 RNPs and ssDNA HDR templates with LNPs edits the chromophore of EGFP to BFP in HEK293T cells. (Upper) Target and donor designs for iGeoCas9-mediated chromophore editing. (Lower) Genome editing efficiencies quantified based on EGFP / BFP signals using iGeoCas9 paired with different sgRNAs ± ssDNA templates. iGeoCas9 RNP with ssDNA generates between 20-40% HDR in HEK293T cells, n = 4 for each group, data are presented as mean values with individual data points. iGeoCas9 used in this figure is NLS-iGeoCas9(C2)-2NLS.

[0021] FIG. 5A-5B LNP-based delivery of iGeoCas9 RNP and ssDNA templates edits endogenous sites of the human genome, a. Genome-editing efficiencies (indels and HDR) by iGeoCas9 paired with different sgRNAs ± ssDNA templates, as quantified by NGS. b. Editing of pathogenic mutations in the CFTR gene through HDR. (Left) Target and donor designs for iGeoCas9-mediated editing of pathogenic mutations. (Right) Genome editing efficiencies quantified by NGS. n = 4 for each group, data are presented as mean values with individual data points. iGeoCas9 used in this figure is NLS-iGeoCas9(C2)-2NLS.

[0022] FIG. 6A-6E Rescreening of ionizable lipids dramatically boosts the delivery efficiency of iGeoCas9 RNP:LNPs assisted by a ssDNA enhancer (enhDNA). a. Schematic diagram of procedures for LNP assembly and general lipid compositions of the two sets of LNP formulations (FX and FC) for ionizable lipid rescreening, b. Screening results indicate that ionizable lipids can dramatically affect the RNP delivery efficiency. Editing assays with tdTom NPCs (with tdTom-g3(23ms)) and HEK293 EGFP cells (with EGFP-g2) were used for the rescreening of FX and FC formulations, respectively. LP01 (IL11) and BP lipid 312 (IL12) were identified as the optimal ionizable lipids for the FX formulation, and lipid III-45 (IL8) was identified as the optimal ionizable lipid for the FC formulation. Cationic formulation (“Cat*”) used ADP- 2k as the pegylated lipid and D-Lin as the ionizable lipid based on the general FC formulation; standard and cationic LNP formulations were assembled under pH 7.0. c. Characterization of microfluidic-formulated LNPs based on FX12 (FX with IL12) and FC8 (FC with IL8) formula. (Upper) Chemical structures of IL12 and IL8. (Lower-left) Cryo-TEM imaging of FX12 and FC8 nanoparticles. (Lower-right) Dynamic light scattering (DLS) shows consistent particle size distribution to Cryo-TEM imaging. The two formulations had good to high encapsulation efficiency for the RNP cargoes andshowed minimal cytotoxicity to cultured cells (NPCs and HEK293 cells), d. FX12 and FC8 formulations show substantially improved efficiency for RNP delivery (with tdTom-g3(23ms) and EGFP-g6(23ms) as the sgRNAs) compared to the standard and cationic formulations with different RNP dosages, even at sub-nM RNP concentrations. Genome editing efficiencies quantified based on tdTom(+) or EGFP(-) signals using iGeoCas9 RNP:LNP complexes, n = 4 for each group, data are presented as mean values with individual data points, e. iGeoCas9 RNP:LNP delivery outcompetes mRNA+sgRNA:LNP delivery, especially with low cargo dosages. mRNA delivery is sensitive to sgRNA stability and requires hyper-modification of sgRNA to enable successful editing at low mRNA / sgRNA dosage, while sgRNA modification does not affect the editing efficiency based on RNP:LNP delivery. iGeoCas9 used in this figure is 2NLS-iGeoCas9(C1)-2NLS.

[0023] FIG. 7A-7I iGeoCas9 RNP:LNPs efficiently edit the liver and lungs of mice. a.Schematic diagram of the experimental design used to evaluate iGeoCas9 RNP:LNP- mediated editing in Ai9 mice. b. Schematic presentation of LNP preparation procedures, c. Modified FX12 LNP formulation (FX12m, with lipid compositions indicated in the table) primarily edits the liver tissue with 37% efficiency. In vivo genome-editing levels in different tissues and different cell types in the liver were quantified by tdTom(+) signals using flow cytometry, d. Modified FC8 LNP formulation (FC8m, with lipid compositions indicated in the table) primarily edits the lung tissue with 16% efficiency. In vivo genome-editing levels in different tissues and different cell types in the lung were quantified by tdTom(+) signals using flow cytometry. For c and d, n = 5 for each group, data are presented as mean values with individual data points and standard deviations; in vitro transcribed sgRNA, tdTom-g3(23), was used, e and f. Nuclei staining with DAPI (blue) and imaging of tdTomato (red) in the edited and non-edited liver or lung tissues, g. sgRNA target designs for PCSK9 and SFTPC gene editing with iGeoCas9 in the liver and lungs, respectively, h and i. In vivo PCSK9 and SFTPC gene editing levels (indels) in the liver and lung tissues using FX12m and FC8m LNP formulations, respectively, as quantified by NGS. n = 5 for each group, data are presented as mean values with individual data points and standard deviations; PBS-only injections are included as negative controls here, and the indels in the liver (FX12m) or in the lungs (FC8m) are shown as the blank editing levels. iGeoCas9 used in this figure is 2NLS-iGeoCas9(C1)-2NLS.

[0024] FIG. 8A-8D Directed evolution of GeoCas9. a. Modelled GeoCas9 structure with mutations highlighted, b. Two rounds of selection to identify improved GeoCas9mutants, c. Mutants and beneficial mutations identified in each round of selection, d. Target cleavage activities of WT-GeoCas9 and R1W1 mutant in the bacterial assay using different spacer and PAM sequences, as reflected by the bacterial survival rates.

[0025] FIG. 9 RNP stability comparison of WT-GeoCas9, GeoCas9(R1 W1), and SpyCas9 using DLS assay. Incubation at 37 °C can gradually lead to SpyCas9 RNP aggregation but does affect WT-GeoCas9 or GeoCas9(R1 W1) within 6 hours.

[0026] FIG. 10A-10B Comparison of iGeoCas9 and SpyCas9 for their genome editing efficiency and RNP stability, a. Gene-editing activity comparison of the two editors using the Ai9 tdTom NPC assay with 10 different sgRNA, respectively (tdTom-g1 to g10 for iGeoCas9, and Spy-tdTom-g1 to g10 for SpyCas9). SpyCas9 and iGeoCas9(C2) showed overall comparable editing level across the 10 targets. NPCs (0.25 M cells in 20 uL buffer) were nucleofected 25 pmol RNP. Editing efficiencies quantified based on the tdTom(+) signal, n = 4 for each group, b. Gene-editing activity comparison of the two editors after incubation at 37 °C for certain time using the HEK293T EGFP assay. Incubation in PBS at 37 °C can gradually lead to function loss for SpyCas9 RNP but does affect iGeoCas9(C2) within 12 hours. Editing efficiencies quantified based on the EGFP(-) signal. EGFP-g6 was used for iGeoCas9(C2). n = 4 for each group, data are presented as mean values with individual data points.

[0027] FIG. 11 Comparison of WT-GeoCas9 and GeoCas9(R1 W1) for their genome editing activities in HEK293T cells to knock down EGFP using different spacer and PAM sequences. GeoCas9(R1W1) shows substantially improved EGFP knock-down efficiency, n = 4 for each group, data are presented as mean values with individual data points.

[0028] FIG. 12A-12B Off-target effect analysis for iGeoCas9-mediated genome editing, a. Schematic illustration of the analysis of on-target and off-target editing activities by iGeoCas9. b. On-target and off-target sequences listed in the tables, editing levels shown in the bar graphs. iGeoCas9 shows overall minimal off-target editing. Editing efficiencies quantified by NGS. n = 2 for each group, data are presented as mean values with individual data points. Target 1 = AAVS1 sitel ; target 2 = AAVS1 site2; target 3 = EMX1 site3.

[0029] FIG. 13A-13C Optimization of LNP formulation for GeoCas9 RNP delivery, a. Comparison of three different genome editors for Ai9 NPC editing based on RNP delivery by LNPs. b. Optimization of the percentage of pegylated lipid ADP-2k in LNP formulations, c. Comparison of different pegylated lipids for their GeoCas9 RNPdelivery efficiency and cytotoxicity with NPCs. n = 4 for each group, data are presented as mean values with individual data points.

[0030] FIG. 14A-14B a. pH-sensitive acetyl linker used in synthetic lipid design, b. Endocytosis pathway in LNP-based delivery promoted by the pH-sensitive acetyl linker in the lipids.

[0031] FIG. 15 Effect of volume ratio (aqueous / organic) and salt concentration on the packaging efficiency of GeoCas9 RNP in LNP. n = 4 for each group, data are presented as mean values with individual data points.

[0032] FIG. 16 Schematic of the whole procedure for LNP-based RNP delivery in cell culture.

[0033] FIG. 17 Co-delivery of GeoCas9 RNPs and ssDNA HDR templates to edit the chromophore of EGFP to BFP in HEK293T cells.

[0034] FIG. 18 Effect of different anionic polymer additives on the packaging efficiency of RNPs in LNPs. n = 4 for each group, data are presented as mean values with individual data points.

[0035] FIG. 19 The effect of cationic lipid, DOTAP, on RNP encapsulation rate under neutral pH.

[0036] FIG. 20A-20D Rescreening of ionizable lipids to improve LNP delivery efficiency of iGeoCas9 RNP. a. Lipid compositions for LNP formulations shown in the tables, b. Structures of ionizable lipids (IL1 to IL13). c. Screening of ionizable lipids for the FX formulation to deliver iGeoCas9 RNP to Ai9 tdTom NPC and HEK293T EGFP cells for genome editing. Genome-editing efficiencies quantified based on tdTom(+) or EGFP(-) signals using iGeoCas9 RNP: LNP complexes in two doses, n = 4 for each group, data are presented as mean values with individual data points, d. Screening of ionizable lipids for the FC formulation to deliver iGeoCas9 RNP to HEK293T EGFP cells for EGFP knock-down. Genome-editing efficiencies quantified based on EGFP(- ) signal using iGeoCas9 RNP: LNP complexes, n = 4 for each group, data are presented as mean values with individual data points.

[0037] FIG. 21 Cryo-EM images of FC8 and FX12 LNPs encapsulating iGeoCas9 RNPs.

[0038] FIG. 22 Comparison of iGeoCas9 and SpyCas9 using the Ai9 tdTom NPC assay based on FX12-LNP delivery of corresponding RNP. LNP characterization shows similar encapsulation properties for iGeoCas9 and SpyCas9 RNP cargoes, but SpyCas9 has much lower efficiency especially at low RNP dosages compared to iGeoCas9. n = 4 for each group, data are presented as mean values with individual data points. Imaging of NPC cultures suggests that SpyCas9 RNP:LNP complexestend to form aggregates in the culture media, probably due to the instability of SpyCasO RNP, while no LNP aggregates were visibly observed for iGeoCasO RNP.

[0039] FIG. 23 Comparison of genome-editing efficiency based on FX12-LNP delivery of mRNA+sgRNA and RNP using the Ai9 tdTom NPC assay. mRNA delivery is sensitive to sgRNA stability and requires hyper-modification of sgRNA to enable successful editing at low mRNA+sgRNA dosage, while sgRNA with modification or not does not affect the editing efficiency based on RNP:LNP delivery, n = 4 for each group, data are presented as mean values with individual data points.

[0040] FIG. 24 Flow analysis examples of genome-editing activities in the whole livers of Ai9 tdTom mice and different cell types in the liver based on the LNP delivery of iGeoCas9 RNPs using FX12m and FC8m formulations. Cell type analysis is based on immunostaining with corresponding fluorescent-labelled antibodies (hepatocyte - CD95+, macrophage - F4 / 80+, and endothelial cells - CD31+).

[0041] FIG. 25 Flow analysis examples of genome-editing activities in the whole lungs of Ai9 tdTom mice and different cell types in the lungs based on the LNP delivery of iGeoCas9 RNPs using FX12m and FC8m formulations. Cell type analysis is based on immunostaining with corresponding fluorescent-labelled antibodies (endothelial cells - CD31+, epithelial cells - CD326+, and immune cells - CD45+).

[0042] FIG. 26 Flow analysis examples of genome-editing activities in different tissues (spleen, kidney and heart) of Ai9 tdTom mice based on the LNP delivery of iGeoCas9 RNPs using FX12m and FC8m formulations.

[0043] FIG. 27A-27C Immune response assessment, a. Levels of different serum cytokines at 6 hours post-injection, including interleukin 2 (IL2), interleukin 6 (IL6), tumor necrosis factor a (TNF-a), and macrophage inflammatory protein 2 (MIP-2). b. Levels of different serum cytokines at 24 hours post-injection, c. Levels of different liver damage enzymes at 2 weeks post-injection, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and transglutaminase 2 (TGM2). Injections of PBS and LPS (lipopolysaccharide, 1 mg / kg) represent negative and positive controls, respectively; RNP-only (RNP / enhDNA) injections are based on an RNP dosage of 4.6 mg / kg; the remaining injections of empty LNP vectors or RNP: LNP complexes follow the lipid or RNP dosages used for the corresponding mouse experiments. Overall, no significant immune response regarding four cytokines and three liver damage enzymes is observed after the injections of RNP, lipids or RNP:LNPs. LPS injections, as positive controls here, induced significant immune responses at early time points, as indicated by the high levels of three cytokines (IL6,MIP2, and TNF-a), but did not exhibit long-term immunogenicity, as indicated by the normal levels of three liver damage enzymes, n = 3 for each group, data are presented as mean values with individual data points and standard deviations.

[0044] FIG. 28 Preliminary results of LNP delivery of prime editor (PE2, based on SpyCas9) to trigger GFP-to-BFP editing in HEK293T cells.

[0045] FIG. 29 Sequences use in the working examples.

[0046] FIG. 30 Primer sequences for NGS amplicon sequencing. All primers for Illumina MiSeq were ordered with (5’-GCTCTTCCGATCT-3’) (SEQ ID NO: xx) at the 5’ end for library preparation and indexing.

[0047] FIG. 31 sgRNA sequences (Alt-R modifications by IDT: 2’-0 methylation and phosphorothioate linkage with the last 3 nucleotides at both 3’- and 5’-ends; m stands for 2’-0 methylation, and * stands for phosphorothioate linkage; spacer). *Note: by IVT - sgRNA prepared by in vitro transcription.

[0048] FIG. 32 ssDNA sequences (HDR templates and enhancer ssDNA with Alt-R modifications by IDT; * stands for phosphorothioate linkage).IV. DEFINITIONS

[0049] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to all forms of nucleic acid (e.g., oligonucleotides) including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA, IncRNA, RNA antagomirs, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), aptamers, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides also include non-coding RNA, which include for example, but are not limited to, RNAi, miRNAs, IncRNAs, RNA antagomirs, aptamers, and any other non-coding RNAs known to those of skill in the art. Polynucleotides include naturally occurring, synthetic, and intentionally altered or modified polynucleotides as well as analogues and derivatives. The term "polynucleotide" also refers to a polymeric form ofnucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof, and is synonymous with nucleic acid sequence. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment as described herein encompassing a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. Polynucleotides can be single, double, or triplex, linear or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0050] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA], In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule: guanine (G) can also base pair with uracil (U). For example, G / U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a guanine (G) is considered complementary to both a uracil (U) and to an adenine (A). For example, when a G / U base-pair can be made at a given nucleotide position of a dsRNA duplex, the position is not considered to be non- complementary, but is instead considered to be complementary.

[0051] Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

[0052] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0053] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul etal., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489), and the like.

[0054] As used herein, a first molecule “specifically binds” or “preferentially binds” or “targets” another molecule if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances, e.g., in a sample, in a cell, etc. In some embodiments, a first molecule “specifically binds” or “targets” if it binds to or associates with the target molecule with an affinity or Ka (that is, an association rate constant of a particular binding interaction with units of 1 / M) of, for example, greater than or equal to about 105M’1. In certain embodiments, the first molecule binds with a Ka greater than or equal to about 106M’1, 107M’1, 108M’1, 109M’1, 101° M’1, 1011M’1, 1012M’1, or 1013M'1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10-5M to 10-13M, or less). In some aspects, specific binding means the targeting moiety binds to the target molecule with a KD of less than or equal to about 10‘5M, less than or equal to about 10'6M, less than or equal to about 10-7M, less than or equal to about 10-8M, or less than or equal to about 10'9M, 10'10M, 10'11M, or 10-12M or less. The binding affinity of a first molecule for a target molecule can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 or BIAcore T200 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like. The term “targets” can also be used to describe complementarity between nucleic acid molecules. As a non-limiting example, a guide RNA that hybridizes with a target sequence of a target nucleic acid (preferentially hybridizes to the target sequence over other sequences) can be said to “target” that target sequence (or target that target nucleic acid).

[0055] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Polypeptides as described herein also include polypeptides having various amino acid additions, deletions, or substitutions relative to the amino acid sequence of a polypeptide of thepresent disclosure. In some embodiments, polypeptides that are homologs of a polypeptide of the present disclosure contain non-conservative changes of certain amino acids relative to the native sequence of a polypeptide of the present disclosure. In some embodiments, polypeptides that are homologs of a polypeptide of the present disclosure contain conservative changes of certain amino acids relative to the native sequence of a polypeptide of the present disclosure, and thus may be referred to as conservatively modified variants. A conservatively modified variant may include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar amino acids are well- known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). A modification of an amino acid to produce a chemically similar amino acid may be referred to as an analogous amino acid.

[0056] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0057] A DNA sequence that "encodes" a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into protein (i.e. , a non-coding RNA (ncRNA) such as, e.g. a tRNA, an rRNA, an siRNA, a microRNA (miRNA), a guide RNA, and the like).

[0058] A "protein coding sequence" or a sequence that encodes a particular protein or polypeptide, is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.

[0059] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., siRNA) or a coding sequence and / or regulate translation of an encoded polypeptide.

[0060] A cell has been “genetically modified” or "transformed" or "transfected" by exogenous DNA or exogenous RNA when such DNA or RNA has been introduced inside the cell. The presence of the exogenous DNA or RNA results in permanent or transient genetic change. Transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0061] “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and / or to prevent the occurrence of the disease or disorder. As an illustrative non-limiting example, in reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[0062] The terms “patient”, “individual”, and “subject” refer to human and non-human subjects, especially mammalian subjects, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0063] The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.

[0064] As used herein the term “isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and / or in which the compound of interest is partially or substantially purified. For example, an "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components present where the substance or a similar substance naturally occurs or from which it is initially prepared. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.

[0065] As used herein, the term “substantially purified” or “purified” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated.

[0066] The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

[0067] The terms "pharmaceutically acceptable", "physiologically acceptable", and “pharmaceutical composition” refer to a biologically acceptable formulation, gaseous,liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition (or simply “pharmaceutical composition”) is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects.

[0068] The phrase a "unit dosage form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, produces a desired effect (e.g., prophylactic or therapeutic effect). In some embodiments, unit dosage forms may be within, for example, ampules and vials, including a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Compositions of the disclosure, including pharmaceutical compositions, can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.

[0069] A "therapeutically effective amount" will fall in a relatively broad range determinable through experimentation and / or clinical trials. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0070] An "effective amount" or "sufficient amount" refers to an amount providing, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a drug), treatments, protocols, or therapeutic regimens agents (including, for example, vaccine regimens), a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured). The doses of an "effective amount" or "sufficient amount" for treatment (e.g., to ameliorate or to provide a therapeutic benefit or improvement) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is also a satisfactory outcome.

[0071] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0072] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0073] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0074] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0075] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0076] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. As such, the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0077] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, it is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0078] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.V. DETAILED DESCRIPTION

[0079] As noted above, provided are lipid nanoparticles (LNPs) formulated for delivery of a payload to a target cell. In some cases, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes: (a) an ionizable lipid (e.g., BP lipid 312, LP01 , and the like) present in a mole percentage of about 36-56% of the total lipids; (b) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 8-20% of the total lipids; (c) cholesterol present in a mole percentage of about 30-50% of the total lipids; (d) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 1- 4% of the total lipids; and (e) a molecular payload. In some cases, the LNP further includes a pegylated cholesterol (e.g., Chol-PEG-2000). In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes: (a) a cationic lipid (e.g., ADC) present in a mole percentage of about 30-55% of the total lipids; (b) an ionizable lipid (e.g., Lipid HI-45, ALC-0315, and the like) present in a mole percentage of about 15-35% of the total lipids; (c) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 8-20% of the total lipids; (d) cholesterol present in a mole percentage of about 10-25% of the total lipids; (e) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 0.8-4% of the total lipids; and (f) a molecular payload.

[0080] Also provided are methods of making and methods of delivering LNPs of the disclosure, e.g., delivery to liver, lung, spleen, kidney, and / or heart tissue of an individual.Compositions and MethodsLipid Nanoparticles (LNPs)

[0081] The present disclosure provides lipid nanoparticles for delivery of a molecular payload (e.g., a nucleic acid payload such as DNA or RNA, e.g., mRNA, and / or a protein payload, e.g., a CRISPR-Cas effector protein such as a Cas9 protein) to cells and tissues. In some cases, the payload includes a ribonucleoprotein (RNP), e.g., a CRISPR-Cas guide RNA complexed with a CRISPR-Cas effector protein. As used herein, the phrase “lipid nanoparticle” (LNP) refers to a transfer vehicle comprising one or more lipids (e.g., ionizable lipids, cationic lipids, non-cationic lipids, neutral lipids, neutral phospholipids, polymerizable lipids, PEG-modified lipids, cholesterol, and the like). In some cases, an LNP includes a molecular payload. In someembodiments, a subject LNP includes an ionizable lipid (e.g., BP lipid 312, LP01 , Lipid HI-45, ALC-0315), a neutral phospholipid (e.g., DOPE), cholesterol, and a pegylated lipid (e.g., DMG-PEG-2000). In some embodiments, the LNP further includes a pegylated cholesterol (e.g., Chol-PEG-2000). In some embodiments, the LNP includes a cationic lipid (e.g., ADC). In some embodiments, the LNP does not include a cationic lipid. In some embodiments, the LNP has a size of less than 125 nm. In some embodiments, the LNP has a size of less than 100 nm.FX12 LNPs

[0082] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (e.g., BP lipid 312, LP01) present in a mole percentage of about 36-56% (e.g., 36-54, 36-52, 36-50, 36-48, 36-47, 38-56, 38-54, 38-52, 38- 50, 38-48, 38-47, 40-56, 40-54, 40-52, 40-50, 40-48, 40-47, 42-56, 42-54, 42-52, 42-50, 42-48, 42-47, 44-56, 44-54, 44-52, 44-50, 44-48, 44-47, 45-56, 45-54, 45-52, 45-50, 45-48, or 45-47) of the total lipids;(b) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 8- 20% (e.g., 8-18, 8-16, 8-14, 8-13, 10-20, 10-18, 10-16, 10-14, 10-13, 11-20, 11- 18, 11-16, 11-14, 11-13, 12-20, 12-18, 12-16, 12-14, or 12-13) of the total lipids;(c) cholesterol present in a mole percentage of about 30-50% (e.g., 32-48, 32- 46, 32-44, 32-42, 32-41 , 34-50, 34-48, 34-46, 34-44, 34-42, 34-41 , 36-50, 36-48, 36-46, 36-44, 36-42, 36-41, 38-50, 38-48, 38-46, 38-44, 38-42, 38-41 , 39-50, 39-48, 39-46, 39-44, 39-42, or 39-41) of the total lipids;(d) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 1-4% (e.g., 1-3, 1-2) of the total lipids; and(e) a molecular payload.

[0083] In some cases, the LNP further includes a pegylated cholesterol (e.g., Chol-PEG- 2000). In some cases, the pegylated cholesterol is present in a mole percentage of about 0.2-1% (e.g., about 0.2-0.6%, 0.3-0.5%, or about 0.4%) of the total lipids.

[0084] In some cases, the subject LNP does not include a cationic lipid.

[0085] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (BP lipid 312) present in a mole percentage of about 36- 56% (e.g., 36-54, 36-52, 36-50, 36-48, 36-47, 38-56, 38-54, 38-52, 38-50, 38- 48, 38-47, 40-56, 40-54, 40-52, 40-50, 40-48, 40-47, 42-56, 42-54, 42-52, 42-50,42-48, 42-47, 44-56, 44-54, 44-52, 44-50, 44-48, 44-47, 45-56, 45-54, 45-52, 45-50, 45-48, or 45-47) of the total lipids;(b) a neutral phospholipid (DOPE) present in a mole percentage of about 8-20% (e.g., 8-18, 8-16, 8-14, 8-13, 10-20, 10-18, 10-16, 10-14, 10-13, 11-20, 11-18, 11-16, 11-14, 11-13, 12-20, 12-18, 12-16, 12-14, or 12-13) of the total lipids;(c) cholesterol present in a mole percentage of about 30-50% (e.g., 32-48, 32- 46, 32-44, 32-42, 32-41 , 34-50, 34-48, 34-46, 34-44, 34-42, 34-41 , 36-50, 36-48, 36-46, 36-44, 36-42, 36-41, 38-50, 38-48, 38-46, 38-44, 38-42, 38-41 , 39-50, 39-48, 39-46, 39-44, 39-42, or 39-41) of the total lipids;(d) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1- 4% (e.g., 1-3, 1-2) of the total lipids; and(e) a molecular payload.

[0086] In some cases, the LNP further includes a pegylated cholesterol (e.g., Chol-PEG- 2000). In some cases, the pegylated cholesterol is present in a mole percentage of about 0.2-1% (e.g., about 0.2-0.6%, 0.3-0.5%, or about 0.4%) of the total lipids.

[0087] In some cases, the subject LNP does not include a cationic lipid.

[0088] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (e.g., BP lipid 312, LP01) present in a mole percentage of about 40-52% (e.g., 40-50, 40-48, 40-47, 42-52, 42-50, 42-48, 42-47, 44-52, 44- 50, 44-48, 44-47, 45-52, 45-50, 45-48, or 45-47) of the total lipids;(b) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 10-15% (e.g., 10-14, 10-13, 11-15, 11-14, 11-13, 12-15, 12-14, or 12-13) of the total lipids;(c) cholesterol present in a mole percentage of about 35-45% (e.g., 36-44, 36- 42, 36-41 , 38-45, 38-44, 38-42, 38-41 , 39-45, 39-44, 39-42, or 39-41) of the total lipids;(d) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 1-2% of the total lipids; and(e) a molecular payload.

[0089] In some cases, the LNP further includes a pegylated cholesterol (e.g., Chol-PEG- 2000). In some cases, the pegylated cholesterol is present in a mole percentage of about 0.2-0.6% (e.g., about 0.3-0.5%, or about 0.4%) of the total lipids.

[0090] In some cases, the subject LNP does not include a cationic lipid.

[0091] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (BP lipid 312) present in a mole percentage of about 40- 52% (e.g., 40-50, 40-48, 40-47, 42-52, 42-50, 42-48, 42-47, 44-52, 44-50, 44- 48, 44-47, 45-52, 45-50, 45-48, or 45-47) of the total lipids;(b) a neutral phospholipid (DOPE) present in a mole percentage of about 10- 15% (e.g., 10-14, 10-13, 11-15, 11-14, 11-13, 12-15, 12-14, or 12-13) of the total lipids;(c) cholesterol present in a mole percentage of about 35-45% (e.g., 36-44, 36- 42, 36-41 , 38-45, 38-44, 38-42, 38-41, 39-45, 39-44, 39-42, or 39-41) of the total lipids;(d) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1- 2% of the total lipids; and(e) a molecular payload.

[0092] In some cases, the LNP further includes a pegylated cholesterol (e.g., Chol-PEG- 2000). In some cases, the pegylated cholesterol is present in a mole percentage of about 0.2-0.6% (e.g., about 0.3-0.5%, or about 0.4%) of the total lipids.

[0093] In some cases, the subject LNP does not include a cationic lipid.

[0094] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (e.g., BP lipid 312, LP01) present in a mole percentage of about 46% of the total lipids;(b) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 12-13% (e.g., about 12.5%) of the total lipids;(c) cholesterol present in a mole percentage of about 40% of the total lipids;(d) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 1-2% (e.g., about 1.5%) of the total lipids; and(e) a molecular payload.

[0095] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (BP lipid 312) present in a mole percentage of about 46% of the total lipids;(b) a neutral phospholipid (DOPE) present in a mole percentage of about 12- 13% (e.g., about 12.5%) of the total lipids;(c) cholesterol present in a mole percentage of about 40% of the total lipids;(d) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1- 2% (e.g., about 1.5%) of the total lipids; and(e) a molecular payload.

[0096] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (e.g., BP lipid 312, LP01) present in a mole percentage of about 46% of the total lipids;(b) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 12-13% (e.g., about 12.4%) of the total lipids;(c) cholesterol present in a mole percentage of about 40% of the total lipids;(d) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 1-2% (e.g., about 1.2%) of the total lipids;(e) a pegylated cholesterol (e.g., Chol-PEG-2000) present in a mole percentage of about 0.3-0.5% (e.g., about 0.4%) of the total lipids; and(f) a molecular payload.

[0097] In some embodiments, a subject LNP (e.g., one for enhanced delivery to liver tissue) includes:(a) an ionizable lipid (BP lipid 312) present in a mole percentage of about 46% of the total lipids;(b) a neutral phospholipid (DOPE) present in a mole percentage of about 12- 13% (e.g., about 12.4%) of the total lipids;(c) cholesterol present in a mole percentage of about 40% of the total lipids;(d) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1- 2% (e.g., about 1.2%) of the total lipids;(e) a pegylated cholesterol (Chol-PEG-2000) present in a mole percentage of about 0.3-0.5% (e.g., about 0.4%) of the total lipids; and(f) a molecular payload.FC8 LNPs

[0098] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes:(a) a cationic lipid (e.g., ADC) present in a mole percentage of about 30-55% (e.g., 30-52, 30-50, 30-49, 30-48, 30-47, 33-55, 33-52, 33-50, 33-49, 33-48, 33- 47, 35-55, 35-52, 35-50, 35-49, 35-48, 35-47, 38-55, 38-52, 38-50, 38-49, 38-48,38-47, 40-55, 40-52, 40-50, 40-49, 40-48, 40-47, 41-55, 41-52, 41-50, 41-49, 41-48, 41-47, 42, 43, 44, 45 or 46) of the total lipids;(b) an ionizable lipid (e.g., Lipid HI-45, ALC-0315) present in a mole percentage of about 15-35% (e.g., 15-33, 15-30, 15-28, 15-27, 18-35, 18-33, 18-30, 18-28, 18-27, 20-35, 20-33, 20-30, 20-28, 20-27, 22-35, 22-33, 22-30, 22-28, 22-27, 23-35, 23-33, 23-30, 23-28, 23-27, 24, 25, or 26) of the total lipids;(c) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 8- 20% (e.g., 8-18, 8-17, 8-16, 10-20, 10-18, 10-17, 10-16, 11-20, 11-18, 11-17, 11-16, 12-20, 12-18, 12-17, 12-16, 12, 13, 14, or 15) of the total lipids;(d) cholesterol present in a mole percentage of about 10-25% (e.g., 10-23, IQ- 20, 10-18, 10-17, 12-25, 12-23, 12-20, 12-18, 12-17, 13-25, 13-23, 13-20, 13-18, 13-17, 14-25, 14-23, 14-20, 14-18, 14-17, 15-25, 15-23, 15-20, 15-18, 15-17, 15, or 16) of the total lipids;(e) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 0.8-4% (e.g., 0.8-3, 0.8-2, 1-4, 1-3, 1-2, 1.2, or 1.5) of the total lipids; and(f) a molecular payload.

[0099] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes:(a) a cationic lipid (e.g., ADC) present in a mole percentage of about 40-48% (e.g., 40-47, 41-48, 41-47, 42, 43, 44, 45 or 46) of the total lipids;(b) an ionizable lipid (e.g., Lipid HI-45, ALC-0315) present in a mole percentage of about 20-30% (e.g., 20-28, 20-27, 22-30, 22-28, 22-27, 23-30, 23-28, 23-27, 24, 25, or 26) of the total lipids;(c) a neutral phospholipid (e.g., DOPE) present in a mole percentage of about 11-16% (e.g., 11-15, 12-16, 11 , 12, 13, 14, 15, or 16) of the total lipids;(d) cholesterol present in a mole percentage of about 14-20% (e.g., 14-18, 14- 17, 15-18, 15-17, 15, or 16) of the total lipids;(e) a pegylated lipid (e.g., DMG-PEG-2000) present in a mole percentage of about 1-3% (e.g., 1-2, 1.2, or 1.5) of the total lipids; and(f) a molecular payload.

[0100] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes:(a) a cationic lipid (ADC) present in a mole percentage of about 42% of the total lipids;(b) an ionizable lipid (Lipid 111-45 or ALC-0315) present in a mole percentage of about 26% of the total lipids;(c) a neutral phospholipid (DOPE) present in a mole percentage of about 15% of the total lipids;(d) cholesterol present in a mole percentage of about 15.8% of the total lipids;(e) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1.2% of the total lipids; and(f) a molecular payload.

[0101] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes:(a) a cationic lipid (ADC) present in a mole percentage of about 42% of the total lipids;(b) an ionizable lipid (Lipid HI-45) present in a mole percentage of about 26% of the total lipids;(c) a neutral phospholipid (DOPE) present in a mole percentage of about 15% of the total lipids;(d) cholesterol present in a mole percentage of about 15.8% of the total lipids;(e) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1.2% of the total lipids; and(f) a molecular payload.

[0102] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes:(a) a cationic lipid (ADC) present in a mole percentage of about 46% of the total lipids;(b) an ionizable lipid (Lipid HI-45 or ALC-0315) present in a mole percentage of about 24% of the total lipids;(c) a neutral phospholipid (DOPE) present in a mole percentage of about 12.5% of the total lipids;(d) cholesterol present in a mole percentage of about 16% of the total lipids;(e) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1.5% of the total lipids; and(f) a molecular payload.

[0103] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes:(a) a cationic lipid (ADC) present in a mole percentage of about 46% of the total lipids;(b) an ionizable lipid (Lipid 111-45) present in a mole percentage of about 24% of the total lipids;(c) a neutral phospholipid (DOPE) present in a mole percentage of about 12.5% of the total lipids;(d) cholesterol present in a mole percentage of about 16% of the total lipids;(e) a pegylated lipid (DMG-PEG-2000) present in a mole percentage of about 1.5% of the total lipids; and(f) a molecular payload.Ionizable lipids

[0104] Ionizable lipids are protonated at low pH, which makes them positively charged, but they remain neutral at physiological pH. The pH-sensitivity of ionizable lipids is beneficial, e.g., for mRNA delivery in vivo, because neutral lipids have less interactions with the anionic membranes of cells and, thus, improve the biocompatibility of lipid nanoparticles. In some cases, ionizable lipids can promote endosome escape and reduce toxicity.

[0105] In some cases, an ionizable lipid of a subject LNP includes a branched tail (e.g., Lipid HI-45, lipid A9, ALC-0315, and the like). Examples of ionizable lipids include, but are not necessarily limited to: BP lipid 312, LP01, Lipid III-45, ALC-0315, lipid A9, D-Lin, DLin-MC3-DMA, ALC-0315, SM-102, LP01, CL1 , TCL053, CKK-E12, and analogs thereof. Other examples include: ATX-002, DLin-DMA, DLenDMA, DLin-D-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin- KC4-DMA, DLin-C2K-DMA, DLin-MP-DMA, DODMA, 98N12-5, C12-200, DLin-C- DAP, DLin-DAC, DLinDAP, DLinAP, D Lin- EG- DMA, DLin-2-DMAP, KL10, KL22, KL25, Octyl-CLinDMA, Octyl-CLinDMA (2R), and Octyl-CLinDMA (2S).

[0106] In some embodiments, a subject LNP includes the ionizable lipid BP lipid 312 or the ionizable lipid LP01 (see, e.g., FX12 LNPs). In some embodiments, a subject LNP includes the ionizable lipid BP lipid 312. In some embodiments, a subject LNP includes the ionizable lipid LP01. In some embodiments, a subject LNP includes the ionizable lipid Lipid HI-45 or the ionizable lipid ALC-0315 (see, e.g., FC8 LNPs). In some embodiments, a subject LNP includes the ionizable lipid Lipid HI-45. In some embodiments, a subject LNP includes the ionizable lipid ALC-0315.

[0107] In some cases, the ionizable lipid (e.g., BP lipid 312 or LP01) is present in a subject LNP in a mole percentage (mole %) of about 36-56% of the total lipids (e.g., 36-54, 36-52, 36-50, 36-48, 36-47, 38-56, 38-54, 38-52, 38-50, 38-48, 38-47, 40-56, 40-54,40-52, 40-50, 40-48, 40-47, 42-56, 42-54, 42-52, 42-50, 42-48, 42-47, 44-56, 44-54,44-52, 44-50, 44-48, 44-47, 45-56, 45-54, 45-52, 45-50, 45-48, or 45-47). In some cases, the ionizable lipid (e.g., BP lipid 312 or LP01) is present in a subject LNP in a mole percentage (mole %) of about 40-52% of the total lipids (e.g., 40-50, 40-48, 40-47, 42-52, 42-50, 42-48, 42-47, 44-52, 44-50, 44-48, 44-47, 45-52, 45-50, 45-48, or45-47). In some cases, the ionizable lipid (e.g., BP lipid 312 or LP01) is present in a subject LNP in a mole percentage (mole %) of about 46% of the total lipids.

[0108] In some cases, the ionizable lipid BP lipid 312 is present in a subject LNP in a mole percentage (mole %) of about 36-56% of the total lipids (e.g., 36-54, 36-52, 36-50, 36-48, 36-47, 38-56, 38-54, 38-52, 38-50, 38-48, 38-47, 40-56, 40-54, 40-52, 40-50, 40- 48, 40-47, 42-56, 42-54, 42-52, 42-50, 42-48, 42-47, 44-56, 44-54, 44-52, 44-50, 44- 48, 44-47, 45-56, 45-54, 45-52, 45-50, 45-48, or 45-47). In some cases, the ionizable lipid BP lipid 312 is present in a subject LNP in a mole percentage (mole %) of about 40-52% of the total lipids (e.g., 40-50, 40-48, 40-47, 42-52, 42-50, 42-48, 42-47, 44- 52, 44-50, 44-48, 44-47, 45-52, 45-50, 45-48, or 45-47). In some cases, the ionizable lipid BP lipid 312 is present in a subject LNP in a mole percentage (mole %) of about 46% of the total lipids.

[0109] In some cases, the ionizable lipid LP01 is present in a subject LNP in a mole percentage (mole %) of about 36-56% of the total lipids (e.g., 36-54, 36-52, 36-50, 36- 48, 36-47, 38-56, 38-54, 38-52, 38-50, 38-48, 38-47, 40-56, 40-54, 40-52, 40-50, 40- 48, 40-47, 42-56, 42-54, 42-52, 42-50, 42-48, 42-47, 44-56, 44-54, 44-52, 44-50, 44- 48, 44-47, 45-56, 45-54, 45-52, 45-50, 45-48, or 45-47). In some cases, the ionizable lipid LP01 is present in a subject LNP in a mole percentage (mole %) of about 40-52% of the total lipids (e.g., 40-50, 40-48, 40-47, 42-52, 42-50, 42-48, 42-47, 44-52, 44-50, 44-48, 44-47, 45-52, 45-50, 45-48, or 45-47). In some cases, the ionizable lipid LP01 is present in a subject LNP in a mole percentage (mole %) of about 46% of the total lipids.

[0110] In some cases, the ionizable lipid (e.g., Lipid HI-45 or ALC-0315) is present in a subject LNP in a mole percentage (mole %) of about 15-35% of the total lipids (e.g., 15-33, 15-30, 15-28, 15-27, 18-35, 18-33, 18-30, 18-28, 18-27, 20-35, 20-33, 20-30, 20-28, 20-27, 22-35, 22-33, 22-30, 22-28, 22-27, 23-35, 23-33, 23-30, 23-28, 23-27, 24, 25, or 26). In some cases, the ionizable lipid (e.g., Lipid HI-45 or ALC-0315) ispresent in a subject LNP in a mole percentage (mole %) of about 20-30% of the total lipids (e.g., 20-28, 20-27, 22-30, 22-28, 22-27, 23-30, 23-28, 23-27, 24, 25, or 26). In some cases, the ionizable lipid (e.g., Lipid 111-45 or ALC-0315) is present in a subject LNP in a mole percentage (mole %) of about 26% of the total). In some cases, the ionizable lipid (e.g., Lipid HI-45 or ALC-0315) is present in a subject LNP in a mole percentage (mole %) of about 24% of the total.

[0111] In some cases, the ionizable lipid Lipid HI-45 is present in a subject LNP in a mole percentage (mole %) of about 15-35% of the total lipids (e.g., 15-33, 15-30, 15-28, 15- 27, 18-35, 18-33, 18-30, 18-28, 18-27, 20-35, 20-33, 20-30, 20-28, 20-27, 22-35, 22- 33, 22-30, 22-28, 22-27, 23-35, 23-33, 23-30, 23-28, 23-27, 24, 25, or 26). In some cases, the ionizable lipid Lipid HI-45 is present in a subject LNP in a mole percentage (mole %) of about 20-30% of the total lipids (e.g., 20-28, 20-27, 22-30, 22-28, 22-27, 23-30, 23-28, 23-27, 24, 25, or 26). In some cases, the ionizable lipid Lipid IH-45 is present in a subject LNP in a mole percentage (mole %) of about 26% of the total lipids. In some cases, the ionizable lipid Lipid IH-45 is present in a subject LNP in a mole percentage (mole %) of about 24% of the total lipids.

[0112] In some cases, the ionizable lipid ALC-0315 is present in a subject LNP in a mole percentage (mole %) of about 15-35% of the total lipids (e.g., 15-33, 15-30, 15-28, 15- 27, 18-35, 18-33, 18-30, 18-28, 18-27, 20-35, 20-33, 20-30, 20-28, 20-27, 22-35, 22- 33, 22-30, 22-28, 22-27, 23-35, 23-33, 23-30, 23-28, 23-27, 24, 25, or 26). In some cases, the ionizable lipid ALC-0315 is present in a subject LNP in a mole percentage (mole %) of about 20-30% of the total lipids (e.g., 20-28, 20-27, 22-30, 22-28, 22-27, 23-30, 23-28, 23-27, 24, 25, or 26). In some cases, the ionizable lipid ALC-0315 is present in a subject LNP in a mole percentage (mole %) of about 26% of the total lipids. In some cases, the ionizable lipid ALC-0315 is present in a subject LNP in a mole percentage (mole %) of about 24% of the total lipids.

[0113] Structure of BP lipid 312

[0114] Structure of LP01Neutral phospholipids

[0117] In some embodiments, a subject LNP includes a neutral phospholipid (e.g., DOPE). In some cases, the neutral phospholipid (e.g., DOPE) is present in a subject LNP in a mole percentage (mole %) of about 8-20% of the total lipids (e.g., 8-18, 8-16, 8-14, 8- 13, 10-20, 10-18, 10-16, 10-14, 10-13, 11-20, 11-18, 11-16, 11-14, 11-13, 12-20, 12- 18, 12-16, 12-14, or 12-13). In some cases, the neutral phospholipid (e.g., DOPE) is present in a subject LNP in a mole percentage (mole %) of about 11-16% of the totallipids (e.g., 11-15, 12-16, 11 , 12, 13, 14, 15, or 16). In some cases, the neutral phospholipid (e.g., DOPE) is present in a subject LNP in a mole percentage (mole %) of about 10-15% of the total lipids (e.g., 10-14, 10-13, 11-15, 11-14, 11-13, 12-15, 12- 14, or 12-13). In some cases, the neutral phospholipid (e.g., DOPE) is present in a subject LNP in a mole percentage (mole %) of about 12-13% of the total lipids. In some cases, the neutral phospholipid (e.g., DOPE) is present in a subject LNP in a mole percentage (mole %) of about 12.5% of the total lipids. In some cases, the neutral phospholipid (e.g., DOPE) is present in a subject LNP in a mole percentage (mole %) of about 15% of the total lipids.(1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)

[0119] Examples of neutral phospholipids include, but are not necessarily limited to: : 5- heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1 ,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).Cholesterol

[0120] In some embodiments, a subject LNP includes cholesterol. In some cases, the cholesterol is present in a subject LNP in a mole percentage (mole %) of about 30- 50% of the total lipids (e.g., 32-48, 32-46, 32-44, 32-42, 32-41 , 34-50, 34-48, 34-46, 34-44, 34-42, 34-41 , 36-50, 36-48, 36-46, 36-44, 36-42, 36-41 , 38-50, 38-48, 38-46,38-44, 38-42, 38-41 , 39-50, 39-48, 39-46, 39-44, 39-42, or 39-41). In some cases, the cholesterol is present in a subject LNP in a mole percentage (mole %) of about 35- 45% of the total lipids (e.g., 36-44, 36-42, 36-41 , 38-45, 38-44, 38-42, 38-41 , 39-45,39-44, 39-42, or 39-41). In some cases, the cholesterol is present in a subject LNP in a mole percentage (mole %) of about 40% of the total lipids.

[0121] In some cases, the cholesterol is present in a subject LNP in a mole percentage (mole %) of about 10-25% of the total lipids (e.g., 10-23, 10-20, 10-18, 10-17, 12-25, 12-23, 12-20, 12-18, 12-17, 13-25, 13-23, 13-20, 13-18, 13-17, 14-25, 14-23, 14-20, 14-18, 14-17, 15-25, 15-23, 15-20, 15-18, 15-17, 15, or 16). In some cases, the cholesterol is present in a subject LNP in a mole percentage (mole %) of about 14-20% of the total lipids (e.g., 14-18, 14-17, 15-18, 15-17, 15, or 16). In some cases, the cholesterol is present in a subject LNP in a mole percentage (mole %) of about 15.8% of the total lipids. In some cases, the cholesterol is present in a subject LNP in a mole percentage (mole %) of about 16% of the total lipids.

[0122] Structure of CholesterolPegylated cholesterol

[0123] In some embodiments, a subject LNP includes a pegylated cholesterol (e.g., Chol- PEG-2000). In some cases, the pegylated cholesterol (e.g., Chol-PEG-2000) is present in a subject LNP in a mole percentage of about 0.2-1% of the total lipids (e.g., 0.2-0.6, 0.3-0.5, or about 0.4). In some cases, the pegylated cholesterol (e.g., Chol- PEG-2000) is present in a subject LNP in a mole percentage of about 0.2-0.6% of the total lipids (e.g., 0.3-0.5, or about 0.4). In some cases, the pegylated cholesterol (e.g.,Chol-PEG-2000) is present in a subject LNP in a mole percentage of about 0.3-0.5% of the total lipids. In some cases, the pegylated cholesterol (e.g., Chol-PEG-2000) is present in a subject LNP in a mole percentage of about 0.4% of the total lipids.In some embodiments, the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.

[0124] Structure of Chol-PEG-2000Pegylated Lipids

[0125] In some embodiments, a subject LNP includes a pegylated lipid (e.g., DMG PEG 2000). In some cases, the pegylated lipid (e.g., DMG PEG 2000) is present in a subject LNP in a mole percentage (mole %) of about 0.8-4% of the total lipids (e.g., 0.8-3, 0.8-2, 1-4, 1-3, 1-2, 1.2, or 1.5). In some cases, the pegylated lipid (e.g., DMG PEG 2000) is present in a subject LNP in a mole percentage (mole %) of about 1-4% of the total lipids (e.g., 1-3, 1-2). In some cases, the pegylated lipid (e.g., DMG PEG 2000) is present in a subject LNP in a mole percentage (mole %) of 1-3% of the total lipids (e.g., 1-2, 1.2, or 1.5). In some cases, the pegylated lipid (e.g., DMG PEG 2000) is present in a subject LNP in a mole percentage (mole %) of 1-2% of the total lipids (e.g., xx). In some cases, the pegylated lipid (e.g., DMG PEG 2000) is present in a subject LNP in a mole percentage (mole %) of about 1.2% of the total lipids. In some cases, the pegylated lipid (e.g., DMG PEG 2000) is present in a subject LNP in a mole percentage (mole %) of about 1 .5% of the total lipids.(1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene)(e.g., 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000)

[0127] PEG-modified lipids (also referred to as pegylated lipids) (e.g., DMG-PEG 2000) include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG- ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1 ,2-diacyloxypropan-3-amines. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG (DMG-PEG), PEG-DLPE, PEG DMPE, PEG- DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid are 1 ,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG- disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG- DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.Cationic lipids

[0128] Cationic lipids typically have a positively charged head group followed by a hydrophobic tail of varying composition. In an aqueous environment, these cationic lipids form micelles with positively charged surfaces that complex with DNA. Examples of cationic lipids include, but are not limited to: ADC, 1 ,2-dioleoyl-3- dimethylammonium-propane (DODAP), 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOPTAC), 1 ,2-dioleoyl-3-(2-(dimethylamino)ethoxy)propylamine (DODEA), and 1 ,2- dimyristoyl-3-trimethylammonium-propane (DMTAP).

[0129] In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes a cationic lipid (e.g., ADC) present in a mole percentage of about 30-55% (e.g., 30-52, 30-50, 30-49, 30-48, 30-47, 33-55, 33-52, 33-50, 33-49, 33-48, 33-47, 35-55, 35-52, 35-50, 35-49, 35-48, 35-47, 38-55, 38-52, 38-50, 38-49, 38-48, 38-47, 40-55, 40-52, 40-50, 40-49, 40-48, 40-47, 41-55, 41-52, 41-50, 41-49, 41-48, 41-47, 42, 43, 44, 45 or 46). In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes a cationic lipid (e.g., ADC) present in a mole percentage of about 40-48% (e.g., 40-47, 41-48, 41-47, 42, 43, 44, 45 or 46). In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes a cationic lipid (e.g., ADC) present in a mole percentage of about 42%. In some embodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes a cationic lipid (e.g., ADC) present in a mole percentage of about 45%. In someembodiments, a subject LNP (e.g., one for enhanced delivery to lung tissue) includes a cationic lipid (e.g., ADC) present in a mole percentage of about 46%.

[0130] For the structure of ADC, see FIG. 2. Also see, e.g., Zhao et al., Nat Nanotechnol. 2024 Aug 23; as well as international patent publication No. WO2024112479, which is incorporated herein by reference in its entirety. Payloads: Nucleic Acid and / or Protein

[0131] LNPs can be used as transfer vehicles for delivery of a molecular payload (e.g., nucleic acid payload such as an RNA, e.g., siRNA, mRNA, guide RNA, or DNA) into a target cell. Examples of molecular payloads that can be encapsulated by a LNP include, but are not necessarily limited to: nucleic acids, proteins (e.g., a therapeutic protein such as an antibody, a CRISPR-Cas effector protein such as a Cas9), polysaccharides, lipids, radioactive substances, therapeutic agents, prodrugs, nutritional supplements, biomarkers, or any combination thereof. In some instances, the payload may comprise more than one type of entity such as a protein (e.g., a gene editing protein such as a Cas9) and a nucleic acid (e.g., a guide RNA).

[0132] In some embodiments, the payload comprises a nucleic acid. Examples of such include, but are not necessarily limited to siRNAs, mRNAs, antisense oligonucleotides, miRs, anti-miRs, shRNAs, expression vectors such as plasmid DNA, DNAs or RNAs encoding a CRISPR-Cas effector protein, CRISPR-Cas guide RNAs, DNAs encoding a CRISPR-Cas guide RNA, or any combination thereof. For example, in some embodiments, the payload includes an RNA (e.g., an mRNA encoding a CRISPR-Cas effector protein. In some embodiments, the payload includes an mRNA (e.g., an mRNA encoding gene of interest, an mRNA encoding a gene editing protein such as a Zinc Finger Nuclease (ZFN), a Transcription Activator-Like Effector Nucleases (TALEN), or a CRISPR-Cas effector protein, and the like). In some embodiments, the payload includes a CRISPR-Cas guide RNA. In some embodiments, the payload includes a CRISPR-Cas guide RNA and an mRNA (e.g., an mRNA encoding a gene editing protein such as CRISPR-Cas effector protein). In some embodiments, the payload includes 2 or more nucleic acids (e.g., two or more siRNAs, two or more mRNAs, two or more guide RNAs, one or more siRNAs plus one or more guide RNAs, one or more siRNAs plus one or more mRNAs, one or more mRNAs plus one or more guide RNAs, one or more siRNAs plus one or more mRNAs plus one or more guide RNA, and the like). In some embodiments, the payload includes a CRISPR-Cas effector protein. In some embodiments, the payload includes a CRISPR-Cas effector protein and a CRISPR-Cas guide RNA, e.g., in some casescomplexed to one another thus forming a ribonucleoprotein (RNP). In some cases, the payload includes a CRISPR-Cas guide RNA and an mRNA encoding a CRISPR- Cas effector protein.

[0133] In some cases, two or more molecular payloads are delivered as part of the same LNP. In some cases, two or more molecular payloads are delivered as part of separate (different) LNPs - and can be delivered simultaneously (as part of the same or different compositions), or delivered serially (one before the other).

[0134] Approaches for designing CRISPR-Cas guide RNAs (also simply referred to herein as “guide RNAs”), and using CRISPR-Cas systems to increase expression or decrease expression of a target gene (e.g., via CRISPRa or CRISPRi, respectively) or to edit a target gene (e.g., induce a mutation in a target gene) are known in the art and any convenient system can be used. For example, when using CRISPRa or CRISPRi to modulate expression of a target gene, a guide RNA is used that hybridizes at or near a transcription start site to inhibit (CRISPRi) or activate (CRISPRa) expression of the target gene. A guide RNA can be said to “target” the gene, and one of ordinary skill in the art would understand how to design an appropriate guide RNA based on the desired outcome - i.e. , they would understand what sequences within the target locus to target.

[0135] In some cases, a molecular payload includes a nucleic acid such as an mRNA or DNA encoding a gene editing protein. In some cases, a molecular payload includes a protein, e.g., a gene editing protein. A gene editing protein may be artificially engineered or may be found in nature. In some embodiments, the gene editing protein is a nuclease that creates a specific break (in some cases one or more singlestranded breaks, i.e., nicks, and in some cases one or more double-stranded breaks (DSBs)) at desired locations in the genome. The cell's endogenous repair mechanisms subsequently repairs the induced break(s) by natural processes, such as homologous recombination (HR) and non-homologous end-joining (NHEJ). Examples of such gene editing proteins include, but are not necessarily limited to: Zinc Finger Nucleases (ZFNs), Transcription Activator- Like Effector Nucleases (TALENs), CRISPR-Cas effector proteins, (e.g., the CRISPR-Cas system), and meganucleases (homing endonucleases).

[0136] Examples of gene editing proteins include, but are not necessarily limited to: a ZFN, a TALEN, a CRISPR-Cas effector protein (which functions in combination with a guide RNA), a meganuclease, a site-specific recombinase, a resolvase / integrase, a transposase, a transposon, and the like. In some cases, the gene editing protein is asite-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb1 integrase, R4 integrase, lambda integrase, HK022 integrase, HP1 integrase, and the like). In some cases, the gene editing protein is a meganulease (homing endonuclease) (e.g., I-Scel, l-Scell, I- Scelll, l-ScelV, l-SceV, l-SceVI, l-SceVII, l-Ceul, l-CeuAIIP, l-Crel, l-CrepsblP, I- CrepsbllP, l-CrepsblllP, l-CrepsblVP, l-Tlil, l-Ppol, Pl-Pspl, F-Scel, F-Scell, F-Suvl, F-Tevl, F-Tevll, l-Amal, l-Anil, l-Chul, l-Cmoel, l-Cpal, l-Cpall, l-Csml, l-Cvul, I- CvuAlP, l-Ddil, l-Ddill, l-Dirl, l-Dmol, l-Hmul, l-Hmull, l-HsNIP, l-Llal, l-Msol, l-Naal, I- Nanl, l-NcllP, l-NgrIP, l-Nitl, l-Njal, l-Nsp236IP, l-Pakl, l-PbolP, l-PculP, l-PcuAI, I- PcuVI, l-PgrIP, l-PoblP, l-Porl, l-PorllP, l-PbpIP, 1-SpBetalP, l-Scal, l-SexlP, l-SnelP, l-Spoml, l-SpomCP, l-SpomlP, l-SpomllP, l-SqulP, l-Ssp6803l, 1-SthPhiJP, I- SthPhiST3P, l-SthPhiSTe3bP, l-TdelP, I-Tevl, l-Tevll, l-Tevlll, l-UarAP, l-UarHGPAIP, l-UarHGPA13P, l-VinIP, l-ZbilP, Pl-Mtul, PI-MtuHIP PI-MtuHIIP, Pl-Pful, Pl-Pfull, Pl- Pkol, Pl-Pkoll, PI-Rma43812IP, PI-SpBetalP, Pl-Scel, Pl-Tful, Pl-Tfull, Pl-Thyl, Pl- Tlil, Pl-Tlil I, and the like). In some cases, the gene editing protein is a resolvase and / or invertase (e.g., Gin, Hin, yb3, Tn3, Sin, Beta, and the like). In some cases, the gene editing protein is a transposon (e.g., bacterial transposons such as Tn3, Tn5, Tn7, Tn9, Tn 10, Tn903, Tn 1681 , and the like; eukaryotic transposons such as Tc1 / mariner super family transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himarl , and the like). In some cases, a gene editing protein is a zinc finger nuclease (ZFN) or a transcription activator like effector nuclease (TALEN).

[0137] With regard to CRISPR-Cas proteins, in class 2 CRISPR systems, the functions of the effector complex (e.g., the cleavage of target DNA) are carried out by a single protein (which can be referred to as a CRISPR-Cas effector protein) - where the natural protein is an endonuclease (e.g., see Zetsche et al, Cell. 2015 Oct 22;163(3):759-71; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97; Shmakov et al., Nat Rev Microbiol. 2017 Mar; 15(3): 169- 182: “Diversity and evolution of class 2 CRISPR-Cas systems”; Koonin et al., Curr Opin Microbiol. 2017 Jun:37:67-78; and Makarova et al., Nat Rev Microbiol. 2020 Feb;18(2):67-83;). As such, the term “class 2 CRISPR-Cas protein” or “CRISPR-Cas effector protein” is used herein to encompass the effector protein from class 2 CRISPR systems - for example, type II CRISPR-Cas proteins (e.g., Cas9), type V CRISPR-Cas proteins (e.g., Cpf1 / Cas12a, C2c1 / Cas12b, C2C3 / Cas12c,Cas12d / CasY, Cas12e / CasX), and type VI CRISPR-Cas proteins (e g., C2c2 / Cas13a, C2C7 / Cas13c, C2c6 / Cas13b). Class 2 CRISPR-Cas effector proteins include type II, type V, and type VI CRISPR-Cas proteins, but the term is also meant to encompass any class 2 CRISPR-Cas protein suitable for binding to a corresponding guide RNA and forming a ribonucleoprotein (RNP) complex.

[0138] A nucleic acid that binds to a class 2 CRISPR-Cas effector protein (e.g., a Cas9 protein; a type V or type VI CRISPR-Cas protein; a Cas12 protein; etc.) (thereby forming a ribonucleoprotein complex (RNP)) and targets the complex to a specific location within a target nucleic acid is referred to herein as a “guide RNA” or “CRISPR-Cas guide nucleic acid” or “CRISPR-Cas guide RNA.” A guide RNA can be used to guide the protein to the target sequence. It is to be understood that in some cases, a hybrid DNA / RNA can be made such that a guide RNA includes DNA bases in addition to RNA bases - but the term “guide RNA” is still used herein to encompass such hybrid molecules.

[0139] A guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a “guide sequence” (also referred to as a “targeting sequence” or a “spacer”), which is a nucleotide sequence that is complementary to (and hybridizes to) a sequence of a target nucleic acid, e.g., a target DNA (and thereby can be said to “target” a specific sequence or “target” a specific gene). The guide sequence can be changed each time a new target sequence is selected. A guide RNA also includes a portion that interacts with (binds to) the CRISPR-Cas effector protein. Because this region does not need to change each time a new target sequence is selected, this region is referred to as a “constant region” or “scaffold” (or “handle”). A guide RNA can be referred to by the protein to which it corresponds. For example, when a CRISPR-Cas effector protein is a Cas9 protein, the corresponding guide RNA can be referred to as a “Cas9 guide RNA.” Likewise, as another example, when a CRISPR-Cas effector protein is a Cas12a protein, the corresponding guide RNA can be referred to as a “Cas12a guide RNA.”

[0140] As will be known to one of ordinary skill in the art, in some embodiments, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” (or tracrRNA and crRNA) and is referred to as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.” In some embodiments, the guide RNA is one molecule (e.g., for some class 2 CRISPR-Cas proteins, the corresponding natural guide RNA is a single molecule; and in some cases, an activator and targeter can be covalently linked to one another, e.g., via interveningnucleotides), and the guide RNA is referred to as a “single guide RNA”, a “singlemolecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.”

[0141] As would be understood to one of ordinary skill in the art, the guide RNA can be introduced into a cell as an RNA (or as a DNA / RNA hybrid) or can be introduced as a nucleic acid encoding the RNA (e.g., a DNA such as an expression vector such as a viral, plasmid, or minicircle DNA), in which case the cell transcribes the RNA from the introduced DNA. In some cases, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., a Pol III promoter such as U6 or H1). In some cases, one or more guide RNAs (e.g., 1, 2, 3, 4, 5, 6, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 2- 10, 2-8, 2-6, 2-5, 2-4, 3-10, 3-8, 3-6, 3-5, two or more, three or more, four or more, or five or more) (or nucleotide sequences that encode said guide RNAs) can be introduced into the same cell (e.g., to target different sequences of the same target nucleic, to target different target nucleic acids, etc.).

[0142] Scaffold sequences for various CRISPR-Cas guide RNAs are known in the art. For example, in some cases, the portion of the targeter-RNA (e.g., crRNA) that contributes to the scaffold (i.e. , is 3’ of the guide sequence) (e.g., when using an S. pyogenes Cas9 protein) includes: 5’-GUUUUAGAGCUAUGCUGUUUUG-3' (SEQ ID NO: 76). In some cases, it includes: 5’-GUUUUAGAGCUA-3' (SEQ ID NO: 77). in some cases, the activator-RNA (e.g., tracrRNA) (e.g., when using an S. pyogenes Cas9 protein) includes: ’5- AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG GCACCGAGUCGGUGCUU-3' (SEQ ID NO: 78). In some cases (e.g., when using an S. pyogenes Cas9 protein) a sgRNA includes 5’- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG-3' (SEQ ID NO: 79). In some cases (e.g., when using an S. pyogenes Cas9 protein) a sgRNA includes 5’- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG AAAAAGUGGCACCGAGUCGGUGCUU-3' (SEQ ID NO: 80). Mutations / variants of the above sequences can also be used and many suitable examples will be known to one of ordinary skill in the art.

[0143] Examples of crRNA repeat sequences (also known as the scaffold) for Cas12a proteins include: LbCas12a crRNA:5’ AAUUUCUACUAAGUGUAGAU 3’ (SEQ ID NO: 81) - [spacer] 3’AsCas12a crRNA:5’ AAUUUCUACUCUUGUAGAU 3’ (SEQ ID NO: 82) - [spacer] 3’ FnCas12a crRNA:5’ AAUUUCUACUGUUGUAGAU 3’ (SEQ ID NO: 83) - [spacer] 3’ PmCas12a crRNA:5’ AAUUUCUACUAUUGUAGAU 3’ (SEQ ID NO: 84) - [spacer] 3’M bCas 12a / M b2Cas 12a / M b3Cas 12a crR NA:5’ AAUUUCUACUGUUUGUAGAU 3’ (SEQ ID NO: 85) - [spacer] 3’ TsCas12a crRNA5’ AAUUUCUACUGUUGUAGAU 3’ (SEQ ID NO: 86) - [spacer] 3’ BsCas12a crRNA5’ AAUUUCUACUAUUGUAGAU 3’ (SEQ ID NO: 87) - [spacer] 3’

[0144] The following sequences are each an example of a scaffold of a naturally existing Cas13a guide RNA (e.g., a scaffold that is 5’ of the guide sequence) (See, e.g., Feng et al., Anal Chem. 2023 Jan 10;95(1):206-217):GUAAGAGACUACCUCUAUAUGAAAGAGGACUAAAAC (SEQ ID NO:88) (Listeria seeligeri) (“Lse”) (LseCas13a)GAUAUAGACCACCCCAAUAUCGAAGGGGACUAAAAC (SEQ ID NO:89) (Leptotrichia shahii) (“Lsh”) (LshCas13a)AUUUAGACCACCCCAAAAAUGAAGGGGACUAAAAC (SEQ ID NO:90) (Leptotrichia buccalis) (“Lbu”) (LbuCas13a)GACCACCCCAAAAAUGAAGGGGACUAAAAC (SEQ ID NO:91) (Leptotrichia buccalis) (“Lbu”) (LbuCas13a)GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC (SEQ ID NO:92) (LwaCas13a)GUCACAACUCCCAUGUAGGCGGAGACUGCAAC (SEQ ID NO:93) (TccCas13a)GGAUUUAGAGUACCCCAAAAAUGAAGGGGACUAAAAC (SEQ ID NO:94) (LtrCas13a)

[0145] In some embodiments, a programmable genome editing protein such as a CRISPR- Cas effector protein (e.g., Cas9, Cas12a, Cas13), a ZF, or TALE is fused to a heterologous protein having any desired activity such as DNA-modifying activity (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity); transcription modulation activity (e.g., fusion to a transcription repressor or transcription activator); an activity that modifies a protein (e.g., a histone) that is associated with target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity). In some such cases the CRISPR- Cas effector protein harbors a mutation that reduces the endogenous nuclease activity (e.g., in some cases renders it a nickase and in some cases renders it catalytically inactive (“dead), e.g., a dCas9). In some cases, a CRISPR-Cas effector protein (e.g., a nickase or ‘dead’ version) is fused to a heterologous protein that has transcription activation activity (e.g., includes a transcription activator domain) and thereby increases transcription (and therefore expression) of a target gene (referred to as CRISPRa). In some cases, a CRISPR-Cas effector protein (e.g., a nickase or ‘dead’ version) is fused to a heterologous protein that has transcription repressor activity (e.g., includes a transcription repression domain) and thereby reduces transcription (and therefore expression) of a target gene (referred to as CRISPRi).

[0146] Examples of proteins (or fragments thereof) that can be used to increase transcription (often referred to as CRISPRa when used with a CRISPR-Cas effector protein) include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP64, VP160, p65 subdomain (e.g., from NFkB), Rta, VPR (which is a fusion of VP64, p65, and Rta), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1 B, MLL1 to 5, ASH1 , SYMD2, NSD1 , and the like; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-ElevenTranslocation (TET) dioxygenase 1 (TET1CD), TET1 , DME, DML1, DML2, ROS1 , and the like. See, e.g., Chavez et al., Nat Methods. 2015 Apr; 12(4): 326-328. In some cases, the CRISPRa system is a SAM system, which includes 3 components that form the DNA-binding complex: (1) a CRISPRa fusion protein (e.g., dCas9 fused to VP64), (2) MS2 aptamer(s) added to the guide RNA (forming a characteristic stem loop structure recognized by MS2), and (3) transcriptional activators P65 (Nuclear Factor NF-KB p65) and HSF1 (Heat Shock Factor 1) fused with an MS2-tag corresponding to the minimal aptamer-binding peptide of the MS2 coat protein. See, e.g., review articles such as Adli, Nat Commun. 2018 May 15;9(1): 1911 ; Becirovic, Cell Mol Life Sci. 2022 Feb 12;79(2):130; and Nidhi S, et al., Int J Mol Sci. 2021 Mar 24;22(7):3327.

[0147] Examples of proteins (or fragments thereof) that can be used as heterologous proteins to decrease transcription (often referred to as CRISPRi when used with a CRISPR-Cas effector protein) include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1 , RIZ1 , and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1 , JMJD2D, JARID1A / RBP2, JARID1B / PLU-1 , JARID1C / SMCX, JARID1 D / SMCY, and the like; histone lysine deacetylases such as HDAC1 , HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1 , SIRT2, HDAC11 , and the like; DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1 , CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0148] In some cases, a programmable genome editing protein such as a CRISPR-Cas effector protein (e.g., Cas9, Cas12a, Cas13), a ZF, a ZFN, or TALE or TALEN is fused to one or more heterologous nuclear localization signals (NLSs). In some cases, 1 to 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 4-10, 4- 9, 4-8, 4-7, 5-10, 5-9, or 5-8 NLSs). In some cases, 2 to 5 NLSs (e.g., 2-4 NLSs, or 2- 3 NLSs). In some cases, about 4 NLSs. In some cases, about 7 NLSs. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 60); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK (SEQ ID NO: 61)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 62) or RQRRNELKRSP (SEQ ID NO: 63); the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 64); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 65) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 66) and PPKKARED (SEQ ID NO: 67) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 68) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 69) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 70) and PKQKKRK (SEQ ID NO: 71) of the influenza virus NS1 ; the sequence RKLKKKIKKL (SEQ ID NO: 72) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 73) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 74) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 75) of the steroid hormone receptors (human) glucocorticoid. In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of a subject protein in the nucleus of a eukaryotic cell. If desired, detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the polypeptide such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.

[0149] In some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) has reduced catalytic activity (e.g., a Cas9 protein with nickase activity or a Cas9 protein that is catalytically inactive, e.g., dCas9). For example, when a Cas9 protein has a mutation at one or more amino acid positions corresponding to D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or a A987 of the Cas9 protein set forth in SEQ ID NO: 2 (e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the variant Cas9 protein can still bind to target DNA in a site-specific manner (because it is still guided to a target DNA sequence by a guide RNA) as long as it retains the ability to interact with the guide RNA. In some cases, Cas9 protein of a subject Cas9 fusion polypeptide is a nickase (e.g., cleaves one strand of a double stranded target nucleic acid but not the other strand) (e.g., the Cas9 protein can be a nickase, e.g., can include one or more amino acid mutations that make it a nickase). For example, in some cases, Cas9 protein of a subject Cas9fusion polypeptide has a mutation in a catalytic domain (e.g., a mutation in a RuvC or HNH domain).

[0150] For example, in some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) can cleave the complementary strand of a target nucleic acid but has reduced ability to cleave the non-complementary strand of a target nucleic acid. For example, the Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some cases, a Cas9 protein has a mutation at residue D10 (e.g., D10A, aspartate to alanine) of SEQ ID NO: 2 (or the corresponding position of any Cas9 protein, e.g., any of the proteins set forth in SEQ ID NOs: 1-36) and can therefore cleave the complementary strand of a double stranded target nucleic acid but has reduced ability to cleave the non-complementary strand of a double stranded target nucleic acid (thus resulting in a single strand break (SSB) instead of a double strand break (DSB) when the variant Cas9 protein cleaves a double stranded target nucleic acid) (see, for example, Jinek et al., Science. 2012 Aug 17;337(6096):816-21). Examples of such amino acid positions in a RuvC domain can include: D10, G12, G17, E762, H982, H983, A984, D986, and / or A987 of the Cas9 protein set forth in SEQ ID NO: 2 (e.g., D10A, G12A, G17A, E762A, H982A, H983A, A984A, and / or D986A).

[0151] In some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) can cleave the non-complementary strand of a target nucleic acid but has reduced ability to cleave the complementary strand of the target nucleic acid. For example, the Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain. Thus, the Cas9 protein can be a nickase that cleaves the non- complementary strand, but does not cleave the complementary strand (e.g., does not cleave a single stranded target nucleic acid). As a non-limiting example, in some embodiments, the Cas9 protein has a mutation at position H840 (e.g., an H840A mutation, histidine to alanine) of SEQ ID NO: 2 (or the corresponding position of any Cas9 protein, e.g., the Cas9 proteins set forth as SEQ ID NOs: 1-36 and can therefore cleave the non-complementary strand of the target nucleic acid but has reduced ability to cleave (e.g., does not cleave) the complementary strand of the target nucleic acid. Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded target nucleic acid). Examples of such amino acid positions in an HNH domain can include: H840, N854, and / or N863 of the Cas9 protein set forth in SEQ ID NO: 2 (e.g., H840A, N854A, and / or N863A).

[0152] In some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target nucleic acid. In some cases, the Cas9 protein is a dCas9 protein. As a non-limiting example, in some cases, the Cas9 protein harbors mutations at residues D10 and H840 (e.g., D10A and H840A) of SEQ ID NO: 2 (or the corresponding residues of another Cas9 protein, e.g., any of the proteins set forth as SEQ ID NOs: 1-36) such that the polypeptide has a reduced ability to cleave (e.g., does not cleave) both the complementary and the non-complementary strands of a target nucleic acid. Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded or double stranded target nucleic acid) but retains the ability to bind a target nucleic acid. For example, a Cas9 protein of a subject Cas9 fusion polypeptide can have a mutation in one or more of amino acid positions in (i) a RuvC domain: corresponding to D10, G12, G17, E762, H982, H983, A984, D986, and / or A987 of the Cas9 protein set forth in SEQ ID NO: 2 (e.g., D10A, G12A, G17A, E762A, H982A, H983A, A984A, and / or D986A); and one or more of amino acid positions in (ii) an HNH domain: corresponding to H840, N854, and / or N863 of the Cas9 protein set forth in SEQ ID NO: 2 (e.g., H840A, N854A, and / or N863A).

[0153] In some cases, a CRISPR-Cas effector protein (e.g., a Cas9 protein) is a variant. In some cases, such a variant is a high fidelity (HF) protein such as a HF Cas9 protein (also referred to as SpCas9-HF1 or HF1 - and also -HF2, -HF3, -HF4) (e.g., see Kleinstiver et al. (2016) Nature 529:490). For example, amino acids N497, R661 , Q695, and Q926 of the amino acid sequence set forth as (SEQ ID NO: 2) (or the corresponding position of another Cas9 protein, e.g., a protein having the amino acid sequence of any of the sequences set forth as SEQ ID NOs: 1-36) can be substituted, e.g., with alanine. In some cases, a suitable parent Cas9 protein exhibits altered PAM specificity. See, e.g., Kleinstiver et al. (2015) Nature 523:481. Additional examples of Cas9 variants that can be used, include, but are not limited to: HiFiCas9 (e.g., R691A), eSpCas9 (e.g., K810A, K1003A, R1060A), eSpCas9 (e.g., D1135E), HypaCas9 (e.g., N692A, M694A, Q695A, H698A), xCas9 (e.g., E108G, S217A, A262T, S409I, E480K, E543D, M694I, E1219V), Sniper-Cas9 (e.g., F539S, M763I, K890N), evoCas9 (e.g., M495V, Y515N, K526E, R661Q), SpartaCas (e.g., D23A, T67L, Y128V, D1251G), LZ3Cas9 (e.g., N690C, T769I, G915M, N980K), miCas9 (e.g., SV40 NLS linker fused with brex27 motif), SuperFi-Cas9 (e.g., Y1010D, Y1013D, Y1016D, V1018D, R1019D, Q1027D, K1031D) (see, e.g., Allemailem et al, Int J Mol Sci. 2023 Apr 11 ;24(8):7052). [relative to SEQ ID NO: 2]

[0154] In some cases, the CRISPR-Cas effector protein is a Cas9. Examples of Cas9 proteins include, but are not limited to, those of SEQ ID NOs: 1-36. In some cases, the CRISPR-Cas effector protein is a Cas12a. Examples of Cas12a proteins include, but are not limited to, those of SEQ ID NOs: 37-49. In some cases, the CRISPR-Cas effector protein is a Cas13. Examples of Cas13 proteins include, but are not limited to, those of SEQ ID NOs: 50-59.

[0155] In some cases, the Cas9 is an iGeoCas9 (see, e.g., international patent publication WO2024112479, which is incorporated herein by reference for such disclosure). For example, in some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to iGeoCas9(C) (SEQ ID NO: 34), which is the same as the wild type GeoCas9 of SEQ ID NO: 1 , but with the following mutations: E149G, T182I, N206D, P466Q, Q817R, E843K, E884G, and K908R (see, e.g., Chen et al., bioRxiv. Preprint. 2023 Nov 15: doi:10.1101 / 2023.11.15.566339). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to iGeoCas9(C) (SEQ ID NO: 34). In some cases, the CRISPR-Cas effector protein comprises the amino acid sequence of SEQ ID NO: 34.

[0156] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to iGeoCas9(G)(SEQ ID NO: 35), which is the same as SEQ ID NO:1 , but with the following mutations: E149G, T182I, N206D, P466Q, E843K, E884G, K908R, T1015A, and D1017N (see, e.g., Chen et al., bioRxiv. Preprint. 2023 Nov 15: doi:10.1101 / 2023.11.15.566339). In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to iGeoCas9(G)(SEQ ID NO: 35). In some cases, the CRISPR-Cas effector protein comprises the amino acid sequence of SEQ ID NO: 35.

[0157] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to ThermoCas9 (R1W1)(SEQ ID NO: 36), which is the same as wild type ThermoCas9 (SEQ ID NO: 6), but includes iGeo-like mutations. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% ormore, 98% or more, 99% or more, or 100%) identical to ThermoCas9 (R1W1)(SEQ ID NO: 36). In some cases, the CRISPR-Cas effector protein comprises the amino acid sequence of SEQ ID NO: 36.

[0158] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the Cas9 sequences of SEQ ID NOs.: 1-36. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the Cas9 sequences of SEQ ID NOs.: 1-36. In some cases, the CRISPR-Cas effector protein comprises the amino acid of any one of SEQ ID NOs.: 1-36.

[0159] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the Cas12a sequences of SEQ ID NOs.: 37-49. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the Cas12a sequences of SEQ ID NOs.: 37-49. In some cases, the CRISPR-Cas effector protein comprises the amino acid sequence of any one of SEQ ID NOs.: 37-49.

[0160] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the Cas13 sequences of SEQ ID NOs.: 50-59. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the Cas13 sequences of SEQ ID NOs.: 50-59. In some cases, the CRISPR-Cas effector protein comprises the amino acid sequence of any one of SEQ ID NOs.: 50-59.

[0161] In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the CRISPR-Cas effector protein sequences of SEQ ID NOs.: 1-59. In some cases, the CRISPR-Cas effector protein comprises an amino acid sequence that is 92% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) identical to any one of the CRISPR-Cas effector protein sequences of SEQ ID NOs.: 1-59. Insome cases, the CRISPR-Cas effector protein comprises the amino acid sequence of any one of SEQ ID NOs.: 1-59.

[0162] For additional information related to programmable gene editing tools (e.g., CRISPR- Cas RNA-guided proteins such as Cas9, CasX, CasY, Cas12a, Cas13, Zinc finger proteins such as Zinc finger nucleases, TALE proteins such as TALENs, CRISPR-Cas guide RNAs, PAMs, and the like) refer to, for example, Dreier, et al., (2001) J Biol Chem 276:29466-78; Dreier, et al., (2000) J Mol Biol 303:489-502; Liu, et al., (2002) J Biol Chem 277:3850-6); Dreier, et al., (2005) J Biol Chem 280:35588-97; Jamieson, et al., (2003) Nature Rev Drug Discov 2:361-8; Durai, et al., (2005) Nucleic Acids Res 33:5978-90; Segal, (2002) Methods 26:76-83; Porteus and Carroll, (2005) Nat Biotechnol 23:967-73; Pabo, et al., (2001) Ann Rev Biochem 70:313-40; Wolfe, et al., (2000) Ann Rev Biophys Biomol Struct 29:183-212; Segal and Barbas, (2001) Curr Opin Biotechnol 12:632-7; Segal, et al., (2003) Biochemistry 42:2137-48; Beerli and Barbas, (2002) Nat Biotechnol 20:135-41; Carroll, et al., (2006) Nature Protocols 1 :1329; Ordiz, et al., (2002) Proc Natl Acad Sci USA 99:13290-5; Guan, et al., (2002) Proc Natl Acad Sci USA 99:13296-301 ; Sanjana et al., Nature Protocols, 7:171-192 (2012); Zetsche et al, Cell. 2015 Oct 22;163(3):759-71 ; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385- 97; Jinek et al., Science. 2012 Aug 17;337(6096):816-21 ; Chylinski et al., RNA Biol. 2013 May; 10(5): 726-37; Ma et al., Biomed Res Int. 2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15644-9; Jinek et al., Elife. 2013;2:e00471 ; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43; Qi et al, Cell. 2013 Feb 28; 152(5): 1173-83; Wang et al., Cell. 2013 May 9;153(4):910-8; Auer et. al., Genome Res. 2013 Oct 31 ; Chen et. al., Nucleic Acids Res. 2013 Nov 1 ;41(20):e19; Cheng et. al., Cell Res. 2013 Oct;23(10):1163-71 ; Cho et. al., Genetics. 2013 Nov; 195(3): 1177-80; DiCarlo et al., Nucleic Acids Res. 2013 Apr;41 (7):4336-43; Dickinson et. al., Nat Methods. 2013 Oct; 10(10): 1028-34; Ebina et. al., Sci Rep. 2013;3:2510; Fujii et. al, Nucleic Acids Res. 2013 Nov 1 ;41(20):e187; Hu et. al., Cell Res. 2013 Nov;23(11):1322-5; Jiang et. al., Nucleic Acids Res. 2013 Nov 1 ;41(20):e188; Larson et. al., Nat Protoc. 2013 Nov;8(11):2180-96; Mali et. at., Nat Methods. 2013 Oct;10(10):957-63; Nakayama et. al., Genesis. 2013 Dec;51(12):835- 43; Ran et. al., Nat Protoc. 2013 Nov;8(11):2281-308; Ran et. al., Cell. 2013 Sep 12; 154(6): 1380-9; Upadhyay et. al., G3 (Bethesda). 2013 Dec 9;3(12):2233-8; Walsh et. al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15514-5; Xie et. al., Mol Plant. 2013 Oct 9; Yang et. al., Cell. 2013 Sep 12; 154(6): 1370-9; Briner et al., Mol Cell.2014 Oct 23;56(2):333-9; Burstein et al., Nature. 2016 Dec 22 - Epub ahead of print; Gao et al., Nat Biotechnol. 2016 Jul 34(7):768-73; Shmakov et al., Nat Rev Microbiol. 2017 Mar;15(3):169-182; Makarova et al., Nat Rev Microbiol. 2020 Feb;18(2):67-83; as well as international patent application publication Nos. W02002099084; WOOO / 42219; WO02 / 42459; W02003062455; W003 / 080809; W005 / 014791 ; W005 / 084190; W008 / 021207; W009 / 042186; WO09 / 054985; and W010 / 065123; U.S. patent application publication Nos. 20030059767, 20030108880, 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919;20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530;20140364333; 20140377868; 20150166983; and 20160208243; and U.S. Patent Nos. 6,140,466; 6,511 ,808; 6,453,242 8,685,737; 8,906,616; 8,895,308; 8,889,418;8,889,356; 8,871 ,445; 8,865,406; 8,795,965; 8,771 ,945; and 8,697,359; all of which are hereby incorporated by reference in their entirety.

[0163] In some cases, a gene editing composition includes a CRISPR-Cas base editor (e.g. Komor et al (2016) Nature. 533(7603):420-424. doi: 10.1038 / nature17946). In some cases, a gene editing composition includes a CRISPR-Cas prime editor (e.g., Anzalone et al (2019) Nature 576: 149-157 https: / / doi.org / 10.1038 / s41586-019-1711- 4).

[0164] In some cases, the CRISPR-Cas effector polypeptide is a Type II CRISPR-Cas effector polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas9 polypeptide. In some cases, the Cas9 polypeptide is a Streptococcus pyogenes Cas9 (spyCas9) polypeptide. In some cases, the Cas9 polypeptide is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the Cas9 polypeptide is a Geobacillus thermodenitrificans Cas9 (saCas9) polypeptide. In some cases, the Cas9 polypeptide is a Neisseria meningitidis Cas9 (saCas9) polypeptide.Methods

[0165] As noted above, the present disclosure provides methods of making / producing subject LNPs, methods of delivering a payload (e.g., protein, DNA, RNA, siRNA, mRNA, guide RNA) to a target cell, and methods of modifying a target DNA.Methods of making / producing

[0166] Methods of making / producing lipid nanoparticles (LNPs) include preparing a lipid mixture by combining the appropriate lipids to form a lipid mixture. In some cases, the appropriate lipids include: the ionizable lipid (e.g., BP lipid 312, LP01), the neutral phospholipid (e.g., DOPE), the cholesterol, and the pegylated lipid (e.g., DMG-PEG- 2000). In some cases, the appropriate lipids also include a pegylated cholesterol (e.g., Chol-PEG-2000). In some cases, the appropriate lipids do not include a cationic lipid. In some cases, the appropriate lipids do not include a cationic lipid. In some cases, the appropriate lipids include: the cationic lipid (e.g., ADC), the ionizable lipid (e.g., Lipid HI-45, ALC-0315), the neutral phospholipid (e.g., DOPE), the cholesterol, and the pegylated lipid (e.g., DMG-PEG-2000). See paragraphs elsewhere herein regarding mole percentages for the lipids - the components of the lipid mixture (including their mole ratios, mole %, etc.) can be as described above with respect to LNPs.

[0167] Methods of making / producing LNPs also include preparing a payload mixture by combining the appropriate molecular payload(s) to form a payload mixture. In some cases, the payload mixture is a ribonucleoprotein (RNP) mixture (e.g., a CRISPR-Cas effector protein such as Cas9, e.g., iGeoCas9, complexed with a guide RNA) - which can be prepared by mixing the protein with the RNA. In some cases, the payload mixture is a nucleic acid mixture (e.g., a guide RNA and an mRNA encoding a CRISPR-Cas effector protein) - which can be prepared by mixing the guide RNA with the mRNA. In some cases, preparation of the payload mixture i.e. , combining the appropriate molecular payload(s) is performed in the presence of a single stranded DNA (ssDNA) enhancer DNA (enhDNA) or an anionic polymer (e.g., poly L-glutamate, e.g., with a molecular weight of 15-50 kDa).

[0168] Methods of making / producing LNPs also include combining, at a pH of about 5 (e.g., pH 4.5-5.5, 4.7-5.3, 4.8-5.2, 4.9-5.1 , or 4.7-5.7), the lipid mixture with the payload mixture (e.g., RNP mixture, nucleic acid mixture), thereby producing the LNP. The combining can take place at any convenient temperature, e.g., room temperature. See, e.g., the experimental examples section below.Methods of delivering a payload

[0169] Also provided are methods of delivering a payload to a target cell (e.g., a eukaryotic cell). Such methods include contacting a target cell with a subject LNP (i.e., an LNP of the present disclosure), thereby delivering the molecular payload into the target cell. In some cases, the cell is in vitro (e.g., is an immortalized cell, e.g., a tissue culture cell line). In some cases, an in vitro cell can be considered ex vivo (e.g., is a primary cell isolated from a patient, has undergone a minimum number of passages, etc.). In some cases, the cell is in vivo (e.g., a subject LNP is administered to an individual and the LNP contacts that target cell in vivo). As such, the present disclosure provides methods of delivering a payload of interest to an individual via administration of a subject LNP.Methods of binding a target nucleic acid

[0170] Also provided are methods of binding (in some cases modifying) a target nucleic acid. Delivering a gene editing protein (e.g., a Zinc Finger Nuclease (ZFN), a Transcription Activator- Like Effector Nucleases (TALEN), a CRISPR-Cas effector protein such as a Cas9, Cas12a, or Cas13, and the like) using a subject LNP finds use in a variety of methods (e.g., a CRISPR-Cas effector protein combination with a guide RNA and in some cases further in combination with a donor template). For example, a CRISPR- Cas effector protein of the present disclosure can be used to (i) modify (e.g., cleave, e.g., nick; methylate; etc.) target nucleic acid (DNA or RNA; single stranded or double stranded); (ii) modulate transcription of a target nucleic acid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid (e.g., for purposes of isolation, labeling, imaging, tracking, etc.); (v) modify a polypeptide (e.g., a histone) associated with a target nucleic acid; and the like. Thus, the present disclosure provides a method of modifying a target nucleic acid. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a CRISPR-Cas effector protein; and b) one or more (e.g., two) guide RNAs - using a subject LNP to deliver (a) and / or (b). In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a CRISPR-Cas effector protein ; b) a guide RNA; and c) a donor nucleic acid (e.g., a donor template) - using a subject LNP to deliver (a), (b), and / or (c). In some cases, the contacting step is carried out in a cell in vitro. In some cases, the contacting step is carried out in a cell in vivo. In some such cases the cell is a lungcell or a liver cell. In some such cases the cell is a liver, lung, spleen, kidney, or heart cell.

[0171] Because a method that uses a CRISPR-Cas effector protein includes binding of the protein to a particular region in a target nucleic acid (by virtue of being targeted there by an associated guide RNA), the methods are generally referred to herein as methods of binding (e.g., a method of binding a target nucleic acid). However, it is to be understood that in some cases, while a method of binding may result in nothing more than binding of the target nucleic acid, in other cases, the method can have different final results (e.g., the method can result in modification of the target nucleic acid, e.g., cleavage / methylation / etc., modulation of transcription from the target nucleic acid; modulation of translation of the target nucleic acid; genome editing; modulation of a protein associated with the target nucleic acid; isolation of the target nucleic acid; etc.).

[0172] For example, the present disclosure provides (but is not limited to) methods of cleaving a target nucleic acid; methods of editing a target nucleic acid; methods of modulating transcription from a target nucleic acid; methods of isolating a target nucleic acid, methods of binding a target nucleic acid, methods of imaging a target nucleic acid, methods of modifying a target nucleic acid, and the like.

[0173] As used herein, the terms / phrases “contact a target nucleic acid” and “contacting a target nucleic acid”, for example, with a CRISPR-Cas effector protein, encompass all methods for contacting the target nucleic acid. For example, a CRISPR-Cas effector protein can be provided to a cell as protein, RNA (encoding the CRISPR-Cas effector protein), or DNA (encoding the CRISPR-Cas effector protein); while a guide RNA can be provided as a guide RNA or as a nucleic acid encoding the guide RNA. As such, when, for example, performing a method in a cell (e.g., inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo), a method that includes contacting the target nucleic acid encompasses the introduction into the cell of any or all of the components in their active / final state (e.g., in the form of a protein(s) for a CRISPR-Cas effector protein; in the form of an RNA in some cases for a guide RNA), and also encompasses the introduction into the cell of one or more nucleic acids encoding one or more of the components (e.g., nucleic acid(s) comprising nucleotide sequence(s) encoding a CRISPR-Cas effector protein , nucleic acid(s) comprising nucleotide sequence(s) encoding guide RNA(s), nucleic acid comprising a nucleotide sequence encoding a donor template, and the like). Because the methods can also be performed in vitro outside of a cell, a method that includes contacting a target nucleicacid, (unless otherwise specified) encompasses contacting outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo, etc.

[0174] In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a CRISPR-Cas effector protein. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a CRISPR-Cas effector protein and a guide RNA. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a CRISPR-Cas effector protein, a first guide RNA, and a second guide RNA. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a CRISPR-Cas effector protein, a guide RNA, and a donor polynucleotide.Target Cells (e.g., for methods of delivering a payload and / or methods of modifying a target nucleic acid)

[0175] A target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome, derived from a chromosome, chromosomal, plasmid, viral, mitochondrial, linear, circular, etc.) and can be from any organism. Suitable target cells for the methods disclosed herein (e.g., methods of delivering a molecular payload, methods of binding a target nucleic acid) include, but are not limited to.

[0176] In some embodiments, a target cell is a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual - in some cases a primary cell, e.g., a primary human cell). A cell can be an in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells (a population of cells). A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. In some cases, the target cell is mammalian cell. In some cases, the target cell is a human cell.

[0177] Target cells can be any convenient cell. In some cases the target cell is a liver, lung, spleen, kidney, or heart cell. In some cases the target cell is a liver or lung cell. In some cases the target cell is a liver cell. In some cases the target cell is a lung cell. Examples of possible target cells include, but are not limited to: a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, an astrocyte, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a liver cell, a lung cell (e.g., a lung epithelial cell), etc. Target cells include stem cells, cancer cells, human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes (e.g., white adipocytes), totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, immune cells (e.g., T cell such as cytotoxic T cell, helper T cell, or regulatory T cell (Treg), B cell, monocyte, natural killer cell, dendritic cell, macrophage, and the like), mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal muscle cells, islet cells (e.g., beta cells), fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells. Target cells include, e.g., lung cells, neurons, astrocytes, islet cells, kidney cells, adipocytes, hepatocytes, endothelial cells, muscle cells, cardiomyocytes, retinal cells, and tissueresident stem cells. Target organs and tissues include, e.g., kidney, liver, bone, pancreas, brain, lung, heart, fat, and the like.Donor Polynucleotide (donor template)

[0178] In some embodiments, a molecular payload that includes a gene editing protein (e.g., a Zinc Finger Nuclease (ZFN), a Transcription Activator- Like Effector Nucleases (TALEN), a CRISPR-Cas effector protein such as a Cas9, Cas12a, or Cas13, and the like) generates site-specific double strand breaks (DSBs) or single strand breaks (SSBs) (e.g., when the protein is a nickase variant) within double-stranded DNA (dsDNA) target nucleic acids, which are repaired either by non-homologous end joining (NHEJ) or homology-directed recombination (HDR).

[0179] In some cases, delivering a gene editing protein such as a CRISPR-Cas effector protein (e.g., a Cas9, Cas12a, or Cas13) (or a nucleic acid encoding same) in combination with a guide RNA (or a nucleic acid encoding same), results in contacting a target DNA with an RNP that includes the protein and the guide RNA, and this can occur under conditions that are permissive for nonhomologous end joining or homology-directed repair. Thus, in some cases, a subject method includes contacting the target DNA with a donor polynucleotide (e.g., by introducing the donor polynucleotide into a cell), wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. In some cases, the method does not comprise contacting a cell with a donor polynucleotide, and the target DNA is modified such that nucleotides within the target DNA are deleted. In some cases, a donor polynucleotide is part of the molecular payload (and therefore is considered part of the LNP - it is delivered by the LNP). In some cases, a donor polynucleotide can be delivered to a cell independent from the LNP (e.g., as part of a different LNP or delivered using a different method (e.g., electroporation, lipofection, injection, viral transduction, and the like).

[0180] In some cases, guide RNA and a CRISPR-Cas protein (e.g., Cas9) are coadministered (e.g., contacted with a target nucleic acid, administered to cells, etc.) with a donor polynucleotide sequence that includes at least a segment with homology to the target DNA sequence, the subject methods may be used to add, i.e. insert or replace, nucleic acid material to a target DNA sequence (e.g. to “knock in” a nucleic acid that encodes for a protein, an siRNA, an miRNA, etc.), to add a tag (e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g. promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), to modify a nucleic acid sequence (e.g., introduce a mutation or to correct a mutation to wild type), and the like. As such, an LNP that includes a CRISPR-Cas protein (or nucleic acid encoding same) and a guide RNA (or nucleic acid encoding same) is useful in any in vitro or in vivo application in which it is desirable to modify DNA (or RNA, e.g., when using Cas13) in a site-specific, i.e. “targeted”, way, for example gene knock-out, gene knock-in, gene editing, gene tagging, etc., as used in, for example, gene therapy, e.g. to treat a disease or as an antiviral, antipathogenic, or anticancer therapeutic, the production of genetically modified organisms in agriculture, the largescale production of proteins by cells for therapeutic, diagnostic, or research purposes, the induction of iPS cells, biological research, the targeting of genes of pathogens for deletion or replacement, etc.

[0181] In applications in which it is desirable to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide comprising a donor sequence to be inserted can also provided to the cell. A donor polynucleotide (also referred to as a donor template) will include a “donor sequence,” which is a nucleic acid sequence to be inserted at the cleavage site induced by a gene editing protein (e.g., a CRISPR-Cas protein). The donor polynucleotide can include sufficient homology to a genomic sequence at the cleavage site, e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the cleavage site, e.g. within about 50 bases or less of the cleavage site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the cleavage site, to support homology-directed repair between it and the genomic sequence to which it bears homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) will support homology- directed repair. Donor sequences can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0182] The donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology- directed repair. In some embodiments, the donor sequence comprises a non- homologous sequence flanked by two regions of homology, such that homology- directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region. Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.

[0183] The donor sequence may comprise certain sequence differences as compared to the genomic sequence, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor sequence at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). In some cases, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e. , changes which do not affect the structure or function of the protein). Alternatively, these sequences differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence. In some cases, the nucleotide difference are intended to change the sequence of the target nucleic acid (e.g., DNA).

[0184] The donor sequence may be provided to the cell as single-stranded DNA, singlestranded RNA (e.g., as part of the guide RNA as used in ‘prime editing’, doublestranded DNA, or double-stranded RNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may 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. (1996) Science 272:886-889. 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 O-methyl ribose or deoxyribose residues. As an alternative to protecting the termini of a linear donor sequence, additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination. A donor sequence 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. Moreover, donor sequences 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). As noted above, donor sequences can be introduced using a subject LNP.

[0185] Because of the presence of a PAM sequence, it is contemplated herein that a CRISPR-Cas effector protein (e.g., Cas9) could potentially cleave inserted sequence either prior to or following homology directed repair (e.g., homologous recombination), resulting in a possible non-homologous-end-joining event and further (e.g., undesired) DNA sequence mutation at a target sequence (e.g., chromosomal locus) of interest. Therefore, to avoid cleavage of the donor sequence before and / or after CRISPR-Cas effector protein mediated homology directed repair, in some embodiments, alternate versions of the donor sequence may be used where mutations (e.g., silent mutations) are introduced. These mutations (e.g., silent mutations) may disrupt CRISPR-Cas effector protein binding and cleavage, but not disrupt the amino acid sequence of the repaired gene. For example, a donor sequence can in some cases include a mutated PAM (e.g., insertion of the donor sequence can result in mutation of the PAM sequence to reduce / eliminate additional cleavage events).Treatment / Administration

[0186] In some embodiments, the present disclosure provides a method of treating a disease, the method comprising administering to an individual in need thereof an effective amount of a subject LNP (comprising an appropriate molecular payload).

[0187] In some cases, LNPs can be administered systemically, regionally or locally, or by any convenient route, for example, by injection, infusion, topically (e.g., transdermally), etc. Possible delivery and administration methods can include parenteral, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intracavity, intracranial, transdermal (topical), transmucosal and rectal administration. Example administration and delivery routes include intravenous, intraperitoneal, intrarterial, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, oral (alimentary), mucosal, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, and intralymphatic. In some cases the delivery route is systemic (e.g., parenteral, intravenous). In some cases, the delivery route is subcutaneous.

[0188] In some cases, a therapeutically effective amount of an LNP is an amount that, when administered to an individual in one or more doses, is effective to slow the progression of a disease or disorder in the individual, or is effective to ameliorate symptoms, or is effective to induce a desirable outcome. For example, atherapeutically effective amount of an LNP can be an amount that, when administered to an individual in one or more doses, is effective to reduce a metric of a symptom of interest, e.g., by at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25%.

[0189] A therapeutic or beneficial effect of treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject A therapeutic or beneficial effect can but need not be complete ablation of all or any particular adverse symptom, disorder, illness, or complication of a disease. Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, or complication caused by or associated with a disease, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, or complications caused by or associated with the disease, over a short or long duration (hours, days, weeks, months, etc.).

[0190] In some cases, a subject method can include steps of evaluating the effectiveness of administration - such evaluation can include, e.g., measuring body weight, oxygen consumption rate, body composition, glucose and fatty acid metabolism parameters, shivering, and any combination thereof.

[0191] Multiple doses of a subject LNP can be administered to an individual in need thereof. Where multiple doses are administered over a period of time, an active agent can be administered once a day, once every other day, once every 3 days, once a week, once every two weeks, once a month, once every 2 months, etc., as needed / desired. The actual frequency of administration, and the actual duration of treatment, depends on various factors. The duration of administration can also vary. As an illustrative example, in some cases, administration can be once a week for 5 weeks, 10 weeks, 15 weeks, 20 weeks, etc.

[0192] The dose to achieve a therapeutic effect, e.g., the dose of molecular payload per kilogram of body weight (mg / kg), will vary based on several factors including, but not limited to: route of administration, the nature of the payload, the amount of payload required to achieve a therapeutic effect, the desired outcome, the specific disease treated, any host immune response to the treatment, the stability of the payload, and the like. One skilled in the art can readily determine an appropriate dose range for administration to an individual. For example, if the payload is an siRNA, the dose will likely range from about 0.1-2 mg / per kilogram (mg / kg) of the weight of the subject(e.g., in some cases about 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg), to achieve a therapeutic effect.

[0193] An effective amount or a sufficient amount can, but need not be, provided in a single administration, may require multiple administrations, and, can but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the need of the subject, type, status and severity of the disease treated or side effects (if any) of treatment. In addition, an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject. Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol.

[0194] An effective amount or a sufficient amount need not be effective in each and every subject treated, or a majority of treated subjects in a given group or population. An effective amount or a sufficient amount means effectiveness or sufficiency in a particular subject, not a group or the general population. As is typical for such methods, some subjects will exhibit a greater response, or less or no response to a given treatment method or use. Thus, appropriate amounts will depend upon the condition treated, the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.).

[0195] With regard to a disease or symptom thereof, or an underlying cellular response, a detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease, or complication caused by or associated with the disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease or disorder.

[0196] Thus, a successful treatment outcome can lead to a "therapeutic effect," or "benefit" of decreasing, reducing, inhibiting, suppressing, limiting, controlling or preventing the occurrence, frequency, severity, progression, or duration of a disease or disorder, or one or more adverse symptoms or underlying causes or consequences of the disease in a subject. Treatment methods and uses affecting one or more underlying causes ofthe disease or adverse symptoms are therefore considered to be beneficial. A decrease or reduction in worsening, such as stabilizing the disease, or an adverse symptom thereof, is also a successful treatment outcome.

[0197] Disclosed methods and uses can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include second actives, such as, biologies (proteins), agents and drugs. Such biologies (proteins), agents, drugs, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any other method or use of the disclosure.

[0198] The compound, agent, drug, treatment or other therapeutic regimen or protocol can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) delivery or administration of a subject LNP as described herein. The disclosure therefore provides combinations where a method or use of the disclosure is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, set forth herein or known to one of skill in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of an LNP as described herein, to a subject.Kits

[0199] Provided are kits / systems for carrying out a subject method. Such kits comprise various combinations of components useful in any of the methods described elsewhere herein.

[0200] A kit can further include one or more additional reagents, where such additional reagents can be any convenient reagent. Components of a subject kit can be in separate containers; or can be combined in a single container. In some cases one or more of a kit’s components are pharmaceutically formulated for administration to a human.

[0201] In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods (e.g., dosing instructions, instructions to administer the component(s) to an individual. The instructions for practicing the subject methods are generally recorded on a suitablerecording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0202] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.1. A lipid nanoparticle (LNP) formulated for delivery of a payload to a target cell, comprising:(a) an ionizable lipid present in a mole percentage of about 36-56% of the total lipids;(b) a neutral phospholipid present in a mole percentage of about 8-20% of the total lipids;(c) cholesterol present in a mole percentage of about 30-50% of the total lipids;(d) a pegylated lipid present in a mole percentage of about 1-4% of the total lipids;(e) a CRISPR-Cas effector protein or an mRNA encoding the CRISPR-Cas effector protein; and(f) a guide RNA. The LNP of 1 , wherein the ionizable lipid is present in a mole percentage of about 40-52% of the total lipids. The LNP of 1 , wherein the ionizable lipid is present in a mole percentage of about 46% of the total lipids. The LNP of any one of 1-3, wherein the ionizable lipid is BP lipid 312 or LP01. The LNP of any one of 1-4, wherein the neutral phospholipid is present in a mole percentage of about 10-15% of the total lipids. The LNP of any one of 1-5, wherein the neutral phospholipid is DOPE. The LNP of any one of 1-6, wherein the cholesterol is present in a mole percentage of about 35-45% of the total lipids. The LNP of any one of 1-6, wherein the cholesterol is present in a mole percentage of about 40% of the total lipids. The LNP of any one of 1-8, wherein the pegylated lipid is present in a mole percentage of about 1.5% of the total lipids. The LNP of any one of 1-8, wherein the pegylated lipid is present in a mole percentage of about 1.2% of the total lipids. The LNP of any one of 1-10, wherein the pegylated lipid is DMG-PEG-2000. The LNP of any one of 1-11 , further comprising a pegylated cholesterol. The LNP of 12, wherein the pegylated cholesterol is present in a mole percentage of about 0.2-1% of the total lipids. The LNP of 12, wherein the pegylated cholesterol is present in a mole percentage of about 0.4% of the total lipids. The LNP of any one of 1-14, wherein the pegylated cholesterol is Chol-PEG-2000. A lipid nanoparticle (LNP) formulated for delivery of a payload to a target cell, comprising:(a) a cationic lipid present in a mole percentage of about 30-55% of the total lipids;(b) an ionizable lipid present in a mole percentage of about 15-35% of the total lipids;(c) a neutral phospholipid present in a mole percentage of about 8-20% of the total lipids;(d) cholesterol present in a mole percentage of about 10-25% of the total lipids;(e) a pegylated lipid present in a mole percentage of about 0.8-4% of the total lipids;(f) a CRISPR-Cas effector protein or an mRNA encoding the CRISPR-Cas effector protein; and(g) a guide RNA. The LNP of 16, wherein the cationic lipid is present in a mole percentage of about 40-48% of the total lipids. The LNP of 16, wherein the cationic lipid is present in a mole percentage of about 42% of the total lipids. The LNP of 16, wherein the cationic lipid is present in a mole percentage of about46% of the total lipids. The LNP of any one of 16-19, wherein the cationic lipid is ADC. The LNP of any one of 16-20, wherein the ionizable lipid is present in a mole percentage of about 20-30% of the total lipids. The LNP of any one of 16-20, wherein the ionizable lipid is present in a mole percentage of about 24% of the total lipids. The LNP of any one of 16-20, wherein the ionizable lipid is present in a mole percentage of about 26% of the total lipids. The LNP of any one of 16-23, wherein the ionizable lipid is Lipid III-45 or ALC-0315. The LNP of any one of 16-24, wherein the neutral phospholipid is present in a mole percentage of about 11-16% of the total lipids. The LNP of any one of 16-24, wherein the neutral phospholipid is present in a mole percentage of about 12% of the total lipids. The LNP of any one of 16-24, wherein the neutral phospholipid is present in a mole percentage of about 15% of the total lipids. The LNP of any one of 16-27, wherein the neutral phospholipid is DOPE. The LNP of any one of 16-28, wherein the cholesterol is present in a mole percentage of about 14-20% of the total lipids. The LNP of any one of 16-28, wherein the cholesterol is present in a mole percentage of about 16% of the total lipids. The LNP of any one of 16-30, wherein the pegylated lipid is present in a mole percentage of about 1-3% of the total lipids. The LNP of any one of 16-30, wherein the pegylated lipid is present in a mole percentage of about 1.5% of the total lipids. The LNP of any one of 16-32, wherein the pegylated lipid is DMG-PEG-2000.34. The LNP of any one of 1-33, wherein the LNP comprises the CRISPR-Cas effector protein complexed with a guide RNA as a ribonucleoprotein (RNP).35. The LNP of any one of 1-34, wherein the CRISPR-Cas effector protein is a Cas9 protein.36. The LNP of any one of 1-34, wherein the CRISPR-Cas effector protein is a Cas9 protein that includes an amino acid sequence that is 80% or more identical to the iGeoCas9 sequence of SEQ ID NO: 34.37. A method of making the LNP of any one of 1-36, the method comprising:(a) combining the CRISPR-Cas effector protein with a guide RNA, thereby forming an ribonucleoprotein (RNP) mixture; or combining the mRNA encoding the CRISPR- Cas effector protein with the guide RNA, thereby forming a nucleic acid mixture;(b) combining the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid to form a lipid mixture; and(c) combining, at a pH of about 5, the lipid mixture with the RNP mixture, or the lipid mixture with the nucleic acid mixture; thereby producing the LNP.38. The method of 37, wherein the combining of step (a) is performed in the presence of a single stranded DNA (ssDNA) enhancer DNA (enhDNA) or anionic polymer, (e.g., poly L-glutamate with a molecular weight of 15-50 kDa).39. The method of 37 or 38, wherein, during the combining of step (b), the cationic lipid is combined with the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid to form the lipid mixture.40. A method of delivering a molecular payload to a eukaryotic cell, the method comprising contacting the eukaryotic cell with the LNP of any one of 1-36, thereby providing the CRISPR-Cas effector protein and a guide RNA to the eukaryotic cell.41. The method of 40, wherein the eukaryotic cell is a liver cell.42. The method of 40, wherein the eukaryotic cell is a lung cell.43. The method of 40, wherein the eukaryotic cell is a spleen, kidney, or heart cell.44. The method of any one of 40-43, wherein the eukaryotic cell is in vitro or ex vivo.45. The method of any one of 40-43, wherein the eukaryotic cell is in vivo.46. The method of 40, wherein the eukaryotic cell is in vivo, wherein the LNP comprises the CRISPR-Cas effector protein complexed with the guide RNA as a ribonucleoprotein (RNP), and wherein said contacting comprises intravenous administration of the LNP to an individual.47. The method of 46, wherein the method results in editing of DNA primarily in cells of the liver.48. The method of 46, wherein the method results in editing of DNA primarily in cells of the lung.EXPERIMENTAL EXAMPLES

[0203] The following examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0204] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0205] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like.Example 1

[0206] Lipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity, and scalable in vivo genome editing if efficacious RNP:LNP complexes can be reliably produced. In the work described here, athermostable Cas9 was engineered from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100X more genome editing of cells and organs compared to the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP:LNP complexes edited a variety of cell types and induced homology-directed repair (HDR) in cells receiving co-delivered single-stranded DNA (ssDNA) templates. Using tissue- selective LNP formulations, genome editing levels of 16-37% efficiency were achieved in the liver and lungs of model reporter mice that received single intravenous injections of iGeoCas9 RNP:LNPs. In addition, iGeoCas9 RNP complexed to biodegradable LNPs edited the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome editing levels observed previously using viral or non-viral delivery strategies. These results show that thermostable Cas9 RNP:LNP complexes are a powerful alternative to mRNA:LNP delivery vehicles and expand the therapeutic potential of genome editing.ResultsDirected evolution of GeoCas9 improves editing efficiency and PAM compatibility

[0207] GeoCas9 is a compact type I l-C CRISPR-Cas9 protein that can function as a robust RNA-guided endonuclease at elevated temperatures (50-65 °C is its optimal temperature range) or in the presence of human plasma. These properties make GeoCas9 an attractive editor for delivery in vivo, particularly in the RNP format. However, GeoCas9 is far less effective than the canonical SpyCas9 at genome editing in mammalian cells and has a more restricted PAM. Wild-type GeoCas9 recognizes a PAM sequence of 5’-N4CRAA-3’ (where R is A / G) and can consequently target a much smaller fraction of the genome than SpyCas9, which has a PAM sequence of 5’-NGG-3’.

[0208] It was rationalized that directed evolution could be used to improve the editing efficiency of GeoCas9 and also minimize its PAM sequence requirement. A bacterial dual-plasmid selection system was used to select for evolved active GeoCas9 variants based on Cas9-mediated cleavage of a plasmid encoding the ccdB toxin gene under the control of an inducible pBAD promoter (Fig. 1a). By changing the Cas9 targets or altering the selection conditions, this targeted degradation of a toxin-encoding plasmid allowed adjustment of the selection pressure to enable directed evolution. To search for a reliable evolutionary starting point with minimal activity in the E. coli assay, 20 different sgRNAs were screened that target the ccdB gene at the protospacers associated with different PAM sequences (Fig. 8) and selection under two sets ofconditions was performed (37 °C or 30 °C for 1.5 hours). Target sequence #6 with a disfavored PAM sequence (ggatGAAA) gave a minimal survival rate under either condition (<0.1% for 30 °C and 2-5% for 37 °C) and was chosen for engineering. Libraries of GeoCas9 mutants were generated by targeting different domains of the protein for random mutagenesis (Fig. 8) and then subjected to the selection system under the conditions at 30 °C. To amplify the most active mutants in these libraries, selected mutants were collected and subjected to another round of selection (Fig. 8). Sequencing of the selected colonies identified frequently appearing beneficial mutations from each library (Fig. 8). For instance, the library targeting BH + Rec domains for random mutagenesis generated mutant GeoCas9(R1) bearing four mutations, E149G, T182I, N206D, and P466Q, which gave >95% survival (vs. <5% with the wild-type protein) in the bacterial assay. The addition of further beneficial mutations identified in the library targeting RuvC + HNH + WED domains, including E843K, K908R, E884G, and Q817R, to the mutant R1 construct produced a new lineage of variant GeoCas9 proteins (Fig. 1 b). Combining a total of eight beneficial mutations yielded a composite mutant, GeoCas9(R1W1), which possesses greatly improved target dsDNA cleavage activity (Fig. 8) and well-preserved thermostability (Tm: 55 °C vs. 60 °C, R1W1 mutant vs. wild-type protein, and 43 °C as the Tmfor wildtype SpyCas9) (Fig. 1c; Figs. 9 and 10).

[0209] The genome editing ability of the engineered GeoCas9 mutants was assessed in neural progenitor cells (NPCs) isolated from Ai9 tdTomato mice. In these cells, successful editing of a stop cassette sequence turns on tdTomato gene expression (Fig. 1 d). Twenty-two sgRNAs were designed to target the SV40-derived poly(A) region using various PAM sequences. RNPs assembled from GeoCas9 mutants and these individual sgRNAs were electroporated into NPCs, and the percentage of tdTomato-positive cells was determined by flow cytometry. The evolved mutants, GeoCas9(R1-GRK) and GeoCas9(R1W1), edited cells with >100-fold greater efficiency relative to the wild-type GeoCas9 with most sgRNAs investigated (Fig. 1e). In addition to editing NPCs, the evolved R1W1 mutant also exhibited robust genome editing in human embryonic kidney (HEK293T) cells and was able to reduce expression of enhanced green fluorescent protein (EGFP) with up to 99% editing efficiency (Fig. 11). These experiments demonstrate that the engineered GeoCas9 mutants can accept a broader range of PAM sequences, including but not limited to 5’-N4CNNA-3’ (vs. wild-type PAM sequences: 5’-N4CRAA-3’) (hereafter GeoCas9(R1-GRK) and GeoCas9(R1W1) are referred to as iGeoCas9(C1) andiGeoCas9(C2) for improved GeoCas9 targeting C-based PAM sequences). Additional editing analysis further establishes that iGeoCas9(C2) is a highly efficient and precise genome editor with minimally detectable off-target effects (Fig. 12).

[0210] To further expand the PAM compatibility of the engineered GeoCas9, the mutations T1015A and D1017N identified from the library targeting the WED + PI domains (Fig. 8) were incorporated into a later variant in the engineering lineage, GeoCas9(R1- GRK), to create GeoCas9(R1WP1) (hereafter referred to as iGeoCas9(G)) that alters the preference of the first base in the essential 4-nt PAM sequence from C to G (Fig. 1f). Taken together, these results show that directed evolution can be used to engineer GeoCas9 for improved genome editing activity and broadened PAM compatibility46. iGeoCas9 RNP formulated in LNPs efficiently edits cells in vitro

[0211] The engineered iGeoCas9s have the potential to induce genome editing in cells and tissues that are not readily editable by other enzymes due to poor stability and / or limited delivery efficiency. To test this, the editing activity of iGeoCas9(C2) was compared to that of two established genome editors, SpyCas9 and iCas12a, an engineered version of LbCas12a45(Figs. 2a, 13). Delivery by RNP nucleofection showed that all three of these enzymes generated robust and similar levels of genome editing in tdTomato NPCs. However, delivery of these RNPs using LNPs led to markedly different results: iGeoCas9 RNP:LNP delivery resulted in >2-fold higher editing efficiency compared to SpyCas9 RNP:LNPs, and iCas12a RNP:LNP delivery did not produce detectable editing in these cells. The improved performance of iGeoCas9 RNP relative to other CRISPR-Cas9 RNPs could be due to its higher stability and, thus, higher specific activity per LNP13. In addition, the larger size of the sgRNA for iGeoCas9 compared to SpyCas9 (139 versus 96 nucleotides) generates an RNP with increased negative charges, which could facilitate LNP encapsulation (Fig. 2a).

[0212] To set up a robust LNP-based system for iGeoCas9 RNP delivery, the lipid formulation was further optimized for RNP encapsulation and LNP assembly. Four commercial lipids were used, including 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (D-Lin), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and two synthetic lipids derived from cholesterol, ADP-2k and ADC, which are newly developed for mRNA delivery47(Fig. 2b). The pegylated lipid, ADP-2k, proved to be key to the successful encapsulation of RNPs into LNPs anddelivery to NPCs (Fig. 13). Low percentages (<1%) of ADP-2k led to relatively large particle sizes, which was not beneficial for LNP stability; on the other hand, high percentages (>5%) of ADP-2k resulted in smaller particle sizes but possibly inhibited the endocytosis processes resulting in reduced editing in NPCs. These observations correspond to the known behaviors of pegylated lipids in enhancing LNP stability, controlling particle size, and regulating circulation time4849.

[0213] It was next examined several pegylated lipids, commercial and synthetic, for their ability to encapsulate and deliver iGeoCas9 RNPs in LNPs (Fig. 13). The commonly used 1 ,2-dimyristoyl-rac-glycerol-methoxy(poly(ethylene glycol)) (DMG-PEG) and other PEG lipids derived from DOPE were found to be less effective in delivering RNPs, meanwhile causing toxicity issues. Interestingly, the synthetic pegylated lipid ADP-2k exhibited minimal toxicity in NPCs, and with its inclusion in LNPs, >90% cell viability was observed. Two dipeptide-fused PEG lipids, Pep-1 k and Pep-2k, also showed high editing levels in NPCs (Figs. 13). The reduced toxicity and enhanced delivery efficiency of these three PEGylated lipids, ADP-2k, Pep-1 k, and Pep-2k, stem from the pH-sensitive, acid-degradable acetal linker used in their synthesis. Specifically, the labile acetal linker is cleaved in the late endosome stage of LNP delivery at a pH of 5-6, which frees the PEG moiety from the lipid molecule to reduce cytotoxicity while destabilizing the endosome to promote RNP release into the cytosol (Fig. 14). Further optimization of other parameters of LNP assembly (including mole and volume ratios of lipids to RNP and salt concentration in the buffer, Fig. 15) established two sets of lipid formulations, a standard formulation (with DOTAP as the cationic lipid) and a cationic formulation (with ADC as the cationic lipid). Both formulations can encapsulate iGeoCas9 RNPs and produce nanoparticles with sizes and polydispersity suitable for cellular delivery (diameter: 170-180 nm, PDI: 0.13-0.17)49 (Fig. 2b).

[0214] The genome editing efficacy of iGeoCas9 RNP: LNP complexes was evaluated in NPCs by targeting the SV40-derived poly(A) stop cassette to turn on tdTomato. iGeoCas9 RNPs were assembled using corresponding sgRNAs and then encapsulated into LNPs of the standard formulation (Fig. 16). Quantification of genome editing by sorting tdTomato-expressing cells after LNP treatment established that LNP-based delivery had comparable delivery efficacy to nucleofection (Fig. 3a). It was next tested whether changes to the sgRNA could further enhance editing efficiency using the LNP delivery strategy. The protospacer region was extended from 21 nt to 23 or 24nt and introduced 2’-0 methylation and phosphorothioate linkages tothe last three nucleotides at both the 5’- and 3’-ends (Fig. 3b). These chemical modifications, known to enhance the chemical stability of the sgRNA18, can also be beneficial to RNP delivery. The LNP strategy was also capable of delivering iGeoCas9 RNPs to HEK293T cells and disrupting the expression of an EGFP transgene with comparable efficiency to that observed using nucleofection (Fig. 3c). The cationic lipid formulation for LNP assembly was found to be slightly more effective for RNP delivery to HEK cells. In addition, the LNP:RNP complexes were stable and maintained high editing efficacy after storage in a neutral buffer (PBS / water 1:1) at 4 °C for over a month (Fig. 3d). Together, these experiments established a robust LNP-based system for delivering iGeoCas9 RNPs to cell lines for genome editing. iGeoCas9 RNPs delivered with ssDNA templates induce site-specific integrations in cells

[0215] It was next tested whether LNPs can co-deliver iGeoCas9 RNPs with a ssDNA template to induce site-specific genomic integrations through homology-directed repair (HDR). The physical features of LNPs that co-package iGeoCas9 RNPs and ssDNA templates 180-200nt in length were first characterized (Fig. 4a). Interestingly, in the presence of ssDNA (with a mole ratio of 1 :1 for RNP:ssDNA), the nanoparticle size was reduced from -180 nm to 140-150 nm. This phenomenon is consistent with a recent study showing that ssDNA helps RNP encapsulation into LNPs and prevents LNP aggregation by transient binding to Cas9 RNPs30.

[0216] It was investigated whether the co-delivery of iGeoCas9 RNPs and ssDNA templates in LNPs could switch EGFP to the blue fluorescent protein (BFP) in a model HEK293T cell line (Fig. 4b). In this cell-based assay, editing of the chromophore Thr-Tyr-Gly in the EGFP transgene by HDR installs a Ser / Thr-His-Gly chromophore and converts EGFP into BFP. Four sgRNAs, rEGFP-R1 to -R4, were designed to target the coding and non-coding strands in the chromophore region for editing. To avoid possible recutting events after incorporation of the desired edits, four ssDNA HDR templates were designed to introduce GFP-to-BFP edits together with additional silent mutations in the DNA sequence. BFP signals were observed with the co-delivery tests based on all 16 combinations of RNPs and ssDNA templates using the standard lipid formulation for LNP assembly. HDR levels, indicated by the percentage of BFP- positive cells, were quantified by flow cytometry and ranged from 20% to 40% and depended on the RNP + ssDNA combinations; non-homologous end-joining (NHEJ) levels were between 50-75%, as shown in Figs. 4b and 17. Interestingly, the HDR experiments produced higher overall editing (HDR + NHEJ) levels compared to EGFPknockdown by RNP only, consistent with the role of ssDNA in promoting RNP encapsulation into LNPs. It was wondered whether other anionic polymers, such as poly-L-glutamate (MW 15-50kDa) and heparin (MW 10-30kDa), could have similar effects (Fig. 18). As expected, the anionic polymer poly-L-glutamate also reduced the LNP size and modestly improved editing levels. However, the addition of heparin resulted in reduced editing, probably due to its inhibitory effect on Cas9 function. These results suggest that anionic polymers promote RNP packaging into LNPs through the charge interaction between the polymer additives and cationic lipids (Fig. 18).

[0217] LNP-based co-delivery of iGeoCas9 RNPs and ssDNA templates was further used to induce HDR at endogenous genomic sites in human cells. Four sets of guide RNAs and corresponding donor ssDNAs were designed to target different sites in the EMX1 gene and AAVS1 locus, respectively, for genome editing based on HDR (Fig. 5a). Both the standard and cationic LNP formulations were evaluated for their ability to deliver editing materials to HEK293T cells. HDR levels were quantified using nextgeneration sequencing (NGS), and LNP:RNP:ssDNA complexes generated up to 66% HDR, with total editing levels up to 95%. This co-delivery system was then applied to cell lines of disease models and tested whether the LNP-based editing materials can correct pathogenic mutations. Cystic fibrosis is a genetic disease caused by mutations in the CFTR gene, which encodes the ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR). Two human bronchial epithelial cell lines (16HBEge) containing nonsense mutations in the CFTR gene, G542X, and W1282X, respectively, were employed for the HDR tests (Fig. 5b). iGeoCas9 RNPs and HDR donors were co-delivered to the HBE cells, resulting in 7% HDR that reverted the pathogenic mutations G542X and W1282X, as quantified by NGS. These results suggest that LNP-based RNP delivery may have therapeutic utility for restorative genome editing in the future.Specific ionizable lipids enable low-dose, efficient iGeoCas9 RNP:LNP-mediated editing

[0218] An additional set of screening experiments were performed to further optimize the iGeoCas9 RNP:LNPs, as the goal was to develop an RNP / LNP formulation with high efficiency and low cytotoxicity / immunogenicity. The standard LNP formulation contains the acid-degradable lipid ADP-2k, which cannot be assembled at acidic pHs, hence requiring the inclusion of the cationic lipid DOTAP (Fig. 19). As DOTAP induces a strong immune response in mice50, LNP formulations lacking DOTAP couldhave significant advantages over the standard formulation. We, therefore, performed a screen to identify LNPs that could encapsulate iGeoCas9 RNPs without DOTAP. Two general formulations, FX and FC, were developed for the LNP screening; they contain an enhancer ssDNA (enhDNA), an ionizable lipid, DMG-PEG instead of ADP- 2k, and were formulated at pH 5.0 (Fig. 6a).

[0219] Thirteen ionizable lipids were evaluated in the FX and FC formulations and were screened for genome editing of tdTomato NPCs and HEK293 EGFP cells, respectively (Fig. 20), using a low RNP dose (5 nM) to identify the most efficient LNP formulations. The lipids, LP01 (IL11) and BP lipid 312 (IL12), were the most effective ionizable lipids identified from the FX formulation screen (Fig. 6b). LP01 is a well- studied biodegradable ionizable lipid that has a half-life of 6 hours in the mouse liver and was previously used for delivering Cas9 mRNA+sgRNA to the liver for genome editing in mice, as demonstrated by Intellia51. The lipid BP 312 is a structural analog of LP01. During the screening of FC formulations, ionizable lipids with branched tails, including ALC-0315 (IL4), lipid A9 (IL5) and lipid HI-45 (IL8), were found to be more effective for RNP delivery than D-Lin (IL1), SM102 (IL2) and L319 (IL6) with linear tails (Fig. 6b), suggesting RNPs with defined three-dimensional structures may impose structural requirements on the encapsulating lipids for effective delivery. Two new formulations, FX12 and FC8, composed of BP lipid 312 (IL12) and lipid III-45 (IL8) based on the general FX and FC formulations, respectively, were thus established.

[0220] Characterization of the FX12 and FC8 LNPs assembled with a microfluidic device demonstrated that both formulations encapsulated RNPs with high efficiency (80% and 98%, respectively) and generated nanoparticles with sizes and polydispersity suitable for in vivo applications (average size: 176 nm and 112 nm, respectively; PDI: 0.10-0.11) (Fig. 6c; and Fig. 21). In addition, FX12 and FC8 LNPs exhibited minimal cytotoxicity and did not impact cell viability in vitro. More importantly, the FX12 and FC8 formulations enabled RNP delivery and genome editing under conditions of ultralow RNP dosages, compared to the previous standard and cationic formulations (Fig. 6d). For instance, FX12 LNPs showed nearly one order of magnitude higher genomeediting activity at a 1 nM RNP dose and over two orders of magnitude higher genomeediting activity at a 100 pM RNP dose, compared to the previously established standard formulation. The new FX12 formulation could also deliver SpyCas9 RNP with improved efficiency but still suffers from the issue of RNP instability (Fig. 22).

[0221] Because LNP formulations similar to FX12 have been used for in vivo Cas9 mRNA+sgRNA delivery51 52, the delivery efficiency of mRNA and RNP by these LNPs were compared (Fig. 6e; and Fig. 23). mRNA+sgRNA:LNP delivery of CRISPR gene editors requires chemical modifications of the sgRNA to prevent its rapid degradation in the cellular environment. Two sets of sgRNAs were therefore evaluated, unmodified (UM, by in vitro transcription) and hyper-modified (HM, by IDT synthesis and PAGE purification), for delivery with iGeoCas9 mRNA and RNP. As expected, the use of HM- sgRNA led to a 3 to > 10-fold improvement in genome-editing activity compared to UM-sgRNA when co-delivered with iGeoCas9 mRNA in LNPs. However, there was little difference between UM and HM-sgRNAs when delivered as RNPs, suggesting the sgRNA is well- protected by the Cas9 protein in the RNP format. In addition, RNP delivery showed higher editing efficiency than mRNA+sgRNA delivery, especially at low doses of editing materials, supporting the conclusion that effective RNP delivery can be more advantageous than mRNA delivery by circumventing inefficient translational processes.LNP-based delivery of IGeoCas9 RNPs edits multiple organs efficiently in vivo

[0222] Having demonstrated that iGeoCas9 RNPs can be delivered by FX12 and FC8 LNPs in vitro with high efficiency, it was then asked if this RNP delivery strategy can trigger in vivo genome editing in mice following intravenous injections. More importantly, it was tested whether iGeoCas9 RNPs can be delivered to organs beyond the liver, which represents a major challenge for LNP-mediated delivery of CRISPR genome editors and other molecular cargoes.

[0223] tdTomato Ai9 mice were employed to assess the delivery and editing efficacy of the LNP-based delivery system for iGeoCas9 RNPs (Fig. 7a). The success of organspecific mRNA delivery using SORT LNPs13prompted us to test the ability of different lipid formulations to deliver genome editors to organs beyond the liver. Small modifications were made to the FX12 and FC8 LNP formulations to afford FX12m and FC8m formulations for in vivo RNP delivery targeting the liver and the lungs, respectively (Figs. 7b to 7d). A single retro-orbital injection of LNPs was performed at an RNP-based dose of 4.6 mg / kg (1.4 mg / kg based on sgRNA) for FX12m LNPs and 2.3 mg / kg (0.7 mg / kg based on sgRNA) for FC8m LNPs. Mice were sacrificed two weeks after LNP injection, and the organs, including the liver, lung, spleen, heart, and kidney, were collected for tdTomato signal analysis to determine genome editing levels (Figs. 7c and 7d). Imaging of the organ slices together with flow quantification of tdTomato-positive cells revealed that iGeoCas9 RNPs could be delivered in vivowith LNPs to induce robust genome editing: the FX12m LNP formulation had an average of 37% editing in the liver and the FC8m formulation generated an average of 16% edits in the lungs (n = 5, controls were PBS-treated Ai9 mice) (Figs. 7c to 7f; and Figs. 24 to 26). Specifically, the FX12m LNP formulation drove the delivery of RNPs primarily to the liver, triggering 34%, 54%, and 75% editing in the hepatocytes, macrophages, and endothelial cells, respectively; the FC8m LNP formulation containing ADC as the biodegradable cationic lipid shifted the delivery specificity to the lungs, generating 41%, 18% and 6% genome edits in endothelial, epithelial, and immune cells, respectively. In addition, genome editing was also observed in other tissues that are challenging delivery targets, such as the heart (1-2% genome editing indicated by the tdTomato signal, Fig. 26). Notably, no detectable immune response was observed in the experimental mice after LNP injection at the given doses of the FX12m or FC8m formulations, suggesting low cytotoxicity and low immunogenicity of the LNP reagents (Fig. 27).

[0224] The high efficacy of the FX12m or FC8m formulations in vivo prompted us to explore their potential for genome editing of the disease-causing genes, PCSK9 and SFTPC, in the liver and lungs, respectively. PCSK9 was selected as the liver gene target because its editing attenuates hypercholesterolemia. The SFTPC gene was selected as the target gene in the lungs because it encodes the surfactant protein C (SFC) and gain-of-function mutations in the gene, such as I73T, can cause interstitial lung diseases53(Fig. 7g). Following similar experimental procedures, LNPs were assembled and injected into wild-type mice. The liver and lung tissues of edited mice 10 days post-injection were collected for NGS analysis to quantify editing levels. The NGS results revealed successful editing of PCSK9 in the liver (with an average of 31 %) (Fig. 7h) and SFTPC in the lungs (with an average of 19%) (Fig. 7i), using the FX12m and FC8m formulations, respectively. Overall, these results highlight the potential of LNP-based RNP delivery system for therapeutic gene editing.Discussion

[0225] Here, a generalizable platform for CRISPR genome editing is described both in vitro and in vivo based on LNP-mediated delivery of a thermostable genome editor in the RNP format. Although RNP delivery offers several potential advantages over viralbased or mRNA-based delivery strategies, its use for in vivo genome editing has been limited to tissue editing based on local administration / injection or liver editing through intravenous injection. RNP delivery usually relies on different nanoparticle materials toencapsulate and transport RNPs; however, their applications for in vivo genome editing are commonly restricted by poor particle uniformity, stability, and biocompatibility. The use of the thermostable iGeoCas9 genome editing enzyme described here, along with newly developed LNP formulations, each independently and combined facilitates robust encapsulation and tissue-selective genome editing in mice. The engineered iGeoCas9 mutants maintain superior stability to the commonly used SpyCas9 under a variety of conditions relevant to in vivo delivery and possess enhanced genome editing capability due to its tolerance of mutations beneficial to function while preserving molecular structure. LNP-mediated RNP delivery (e.g., iGeoCas9 RNP delivery) may provide a new approach to targeted in vivo genetic treatments.

[0226] The LNP-assisted RNP delivery described here generated genome edits in vivo in both the mouse liver and lungs, depending on the LNP formulation used. The delivery specificity could be regulated by the electrostatic charge properties of the LNPs. In particular, LNPs prepared with the biodegradable cationic lipid ADC targeted the lungs preferentially compared to the liver. This shift in targeting preference is hypothesized to involve differential recruitment of plasma proteins to the LNP surface, which changes the LNP cell tropism by altering the cell surface receptors they engage in vivo. LNPs with different lipid compositions or formulated under different conditions (e.g., solvent pH, manual vs microfluidic mixing, and others) would lead to different surface properties of the nanoparticles and induce tunable delivery specificity. Together, these findings demonstrate the utility of RNP: LNPs for both ex vivo and in vivo genome editing in tissues in addition to the liver and suggest they have great potential for extending applications of CRISPR-Cas genomic therapies.Materials and Methods Ethical statement

[0227] The research presented here complies with all relevant ethical regulations. All experiments involving animals were reviewed and approved by the Animal Care and Use Committee (ACUC) at the University of California, Berkeley, prior to commencing the study.Plasmid construction

[0228] Plasmids used for the expression of different Cas proteins in this study were built based on a pCold vector. The inserts encoding Cas proteins contain an N-terminal CL7 tag followed by an HRV-3C protease cleavage site and a C-terminal His6tag following another HRV-3C protease cleavage sequence. The insert for the final NLS-GeoCas9(R1W1)-2NLS protein contains an N-terminal sequence consisting of different tags, His6-CL7-MBP (MBP: maltose-binding protein) followed by an HRV-3C protease cleavage site. The cloning reactions were carried out in a 50-pl reaction containing 1 ng of template plasmid, 1.25 pl of 10 mM dNTP, and 1.25 pl of 10 pM each primer using Phusion high-fidelity DNA polymerase (NEB, CAT# M0530L). After PCR, the reactions were treated with 1 pl of Dpnl (NEB, CAT# R0176L) for 1 hour at 37 °C before gel purification. The plasmids were ligated based on Gibson assembly (NEB master mix, CAT# E2611 L) of the plasmid backbone and insert sequences. The sequences of all the plasmid constructs were confirmed via full plasmid sequencing (Primordium).Nucleic acid preparation

[0229] All the DNA and RNA oligos used in this study, unless otherwise indicated, were purchased from Integrated DNA Technologies, Inc. (IDT) and passed the quality control standard set by IDT. The hyper-modified sgRNAs used in the study were laboratory-purified by polyacrylamide gel electrophoresis (PAGE). Some of the sgRNAs and ssDNA HDR templates purchased from IDT possess chemical modifications at 3’- or 5’-ends. The mRNA encoding 2NLS-iGeoCas9(C1)-2NLS was purchased from TriLink and purified with a silica membrane.Lipid material preparation

[0230] Commercial lipid materials used in this study were purchased from BroadPharm, Inc., Avanti Polar Lipids, Inc., and Cayman Chemical Co, Inc. Acid-degradable lipids, ADP and ADC, were laboratory-synthesized following the procedures in our previous publication47.In vitro transcription of sgRNA

[0231] Four sgRNAs (UM-tdTom-g3, UM-tdTom-g7, UM-gPCSK9, and UM-gSFTPC; UM stands for “unmodified”) used in this study were prepared in milligram scale through in vitro transcription (IVT) using HISCRIBE T7 High Yield RNA Synthesis Kit (NEB, CAT# E2040S). Following the general protocol provided by the supplier, each IVT reaction (1.2-1.4 mL) uses one RNA-synthesis kit together with a dsDNA template (30-50 ug) encoding the sgRNA sequence under a T7 promoter. The IVT reaction mixture was incubated at 37 °C for 10-12 hours, then treated with DNAse I (100 units, NEB, CAT# M0303S) and incubated for another 3-4 hours before being quenched by a 2X STOP solution containing formamide, bromophenol blue, xylene cyanol, and EDTA. Urea-PAGE was used for sgRNA purification, and the gel fraction containing the desired sgRNA was crushed into fine pieces and subjected to RNA extraction at4°C overnight using sodium acetate buffer (300 mM, pH 5.0). The extracted sgRNA was concentrated using Amicon Ultra Centrifugal Filter (10-kDa cutoff) to a total volume of 3-5 mL, and the concentrated RNA solution was treated with 10 mL cold isopropanol to allow the sgRNA to precipitate at -20°C over 6 hours. The sgRNA was pelleted by centrifuge and washed using cold 70% ethanol three times. The isolated sgRNA was further dissolved in 1X RCUTSMART buffer (1 mL, NEB, CAT# B6004S), subjected to terminal triphosphate removal using calf intestinal alkaline phosphatase (CIP, 100 units, NEB, CAT# M0525S) and incubated at 37 °C for 6 hours. The reaction mixture was then diluted with sodium acetate buffer (4 mL, 300 mM, pH 5.0) and subjected to phenol / chloroform (5 mL, saturated, pH 5-6) extraction by vigorous vortex and centrifuge; the aqueous phase was further washed with chloroform (5 mL) by vigorous vortex and centrifuge three times. The sgRNA-containing aqueous solution was finally subjected to RNA precipitation and isolation; the pellet was dried in the open air to give purified sgRNA.

[0232] Purified IVT-sgRNAs were dissolved in an endotoxin-free storage buffer (500 uL; 25 mM NaPi, 150 mM NaCI, and 200 mM trehalose at pH 7.50). sgRNAs were reannealed by incubation at 64 °C for 5 minutes, followed by gradual cooling to room temperature. The sgRNA concentration was Nanodrop-determined (after 10-50X dilution). The final yields of GeoCas9 sgRNA by IVT: UM-tdTom-g3 and g7, 4~5 mg / reaction; UM-gPCSK9 and UM-gSFTPC, 8~12 mg / reaction.Directed evolution of GeoCas9

[0233] A chloramphenicol-resistant (CAM+) bacterial expression plasmid was built to have the insert gene of GeoCas9 together with its corresponding sgRNA that targets the ccdB gene in the selection plasmid with a PAM of GAAA (g6). Libraries of GeoCas9 mutants were generated by error-prone PCR to introduce random mutagenesis in three different regions (BH-Rec, RuvC-HNH-WED, and WED-PI). The error-prone PCR (with an error rate of 3- to 5-nucleotide mutations per kilobase) was carried out with the Taq DNA polymerase (NEB, CAT# M0273S) in a reaction containing 2 pl of 10 mM primers, 1.5 pl of 10 mM MnCl2, 2 ng of template plasmid. The plasmid libraries were generated by ligating the mutated fragments with the remaining part of the plasmid through Gibson assembly. The plasmid libraries (~100 ng DNA after clean-up) were electroporated into 50 pl of electronically competent cells made from E. coli strain BW25141(DE3) that contains the selection plasmid encoding the arabinose-inducible ccdB toxin gene. After recovery of the electroporated bacteria in 750 pl of SOB for 1.5 hours at 30 °C, the bacteria culture was concentrated; 1% of thetotal culture was plated onto a Petri agar dish containing only CAM (as control), and the remainder culture was plated on another Petri agar-dish containing both arabinose and CAM. Positive colonies that grew on the plates containing both arabinose and CAM were collected in a pool, retransformed (with ~2 ng plasmid), and replated (100 pl of transformed culture on both control and selection plates). Plasmids of individual colonies from the replated plate were sequenced to obtain mutational information. Validation of the positive clones in the bacterial assay followed the same procedure.Protein expression and purification

[0234] All the proteins in this study were expressed in E. coli BL21 (DE3) cells (Sigma- Aldrich) cultured in 2x YT medium supplemented with the antibiotics of ampicillin. The cultivation was carried out at 37 °C with a shaking speed of 160 rpm after inoculation with an overnight starter culture in LB medium containing ampicillin at a ratio of 1:40. When the optical density (OD6oo) of the culture reached 0.8-0.9, the culture was cooled down to 4 °C on ice. The expression of Cas proteins was induced by the addition of isopropyl p-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM and incubated at 15.8-16 °C with a shaking speed of 120 rpm for 14-16 hours.

[0235] To purify the Cas (or fusion) proteins, the cultured cells were harvested and resuspended in lysis buffer (50 mM Tris-HCI, 20 mM imidazole, 1.2 M NaCI, 10% (v / v) glycerol, 1 mM TCEP, 0.5 mM, and COMPLETE protease inhibitor cocktail tablets (Millipore Sigma, 1 tablet per 50 ml) at pH 7.5), disrupted by sonication and centrifuged at 35,000 xg for 45 min. Ni-NTA resin was treated with the supernatant at 4 °C for 60 min, washed with wash buffer-1 (lysis buffer without protease inhibitor cocktail tablet), and eluted with elution buffer (50 mM Tris-HCI, 300 mM imidazole, 1.2 M NaCI, 10% (v / v) glycerol, and 1 mM TCEP at pH 7.5) to give crude His-tagged Cas proteins. The nickel elution was then subjected to Im7-6B resin in a slow gravity column repeatedly (3-4 times). The Im7-6B resin was washed with wash buffer-2 (50 mM Tris-HCI, 1.2 M NaCI, 10% (v / v) glycerol, and 1 mM TCEP at pH 7.5) before being treated with HRV-3C protease (1 % weight to crude Cas protein) for 2-2.5 hours to release the Cas proteins from the CL7 and His6tags. Heparin affinity column was used to further purify the desired proteins. The protein fractions were collected, concentrated, and stored in the storage buffer (25 mM NaPi, 150 mM NaCI, and 200 mM trehalose at pH 7.50) after buffer exchange. The final yields of different Cas proteins (all with two copies of NLS at both N- and C-termini): wild-type GeoCas9, ~10 mg per 1 L culture; GeoCas9 mutants, in a range of 2-10 mg per 1 L culture;SpyCas9, ~4 mg per 1 L culture; iCas12a, ~30 mg per 1 L culture; PE2 (nSpyCas9- RT), 1-2 mg per 1 L culture.

[0236] The purification of NLS-GeoCas9(R1W1)-2NLS and 2NLS-GeoCas9(R1-GRK)-2NLS proteins is slightly different after Ni-NTA resin purification. The nickel elution was subjected to dialysis against dialysis buffer (50 mM Tris-HCI, 10 mM imidazole, 1.2 M NaCI, 10% (v / v) glycerol, and 1 mM TCEP at pH 7.5) containing HRV-3C protease (1% weight to crude Cas protein) for 12-15 hours. The tag-cleaved protein was then loaded to a heparin column and washed with 80 column volumes of buffer containing 0.1% Triton X-114 at 4 °C to minimize endotoxin impurities. The protein fractions were collected, concentrated, and subjected to further purification using a size-exclusion column in an endotoxin-free manner. The purified protein was stored in an endotoxin- free storage buffer (25 mM NaPi, 150 mM NaCI, and 200 mM trehalose at pH 7.50). The final yield of the desired GeoCas9 mutant: 3-5 mg per 1 L culture.Measurement of protein melting temperatures

[0237] Protein melting temperatures were measured using the thermal shift assay (GloMelt, #33021). The assay was performed on a quantitative PCR system with a temperature increase rate of 2 °C / min. The protein melting temperatures were determined as the peak values in the derivative curves of the melting curves.Cell lines and culture conditions

[0238] NPCs were isolated from embryonic day 13.5 Ai9-tdTomato homozygous mouse brains. Cells were cultured as neurospheres at 37 °C with 5% CO2 in NPC medium: DMEM / F12 (Gibco, CAT# 10565018) with GLUTAMAX supplement, sodium pyruvate, 10 mM HEPES, nonessential amino acid (Gibco, CAT# 11140076), penicillin and streptomycin (Gibco, CAT# 10378016), 2-mercaptoethanol (Gibco, CAT# 21985023), B-27 without vitamin A (Gibco, CAT# 12587010), N2 supplement (Gibco, CAT# 17502048), and growth factors, bFGF (BioLegand, CAT# 579606) and EGF (Gibco, CAT# PHG0311) (both 20 ng / ml as final concentration). NPCs were passaged using the MACS Neural Dissociation Kit (Papain, CAT# 130-092-628) following the manufacturer’s protocol. bFGF and EGF were refreshed every three days, and cells were passaged every 4-5 days. Pre-coating with a coating solution containing poly- DL-ornithine hydrobromide (Sigma-Aldrich, CAT# P8638), laminin (Sigma-Aldrich, CAT# 11243217001), fibronectin bovine plasma (Sigma-Aldrich, CAT# F4759) was required for culturing cells in 96-well plates.

[0239] HEK293T and HEK293T-EGFP cells were grown in a medium containing DMEM (Gibco, CAT# 10569010), high glucose, GLUTAMAX supplement, sodium pyruvate,10% FBS, and penicillin and streptomycin (Gibco, CAT# 10378016) at 37 °C with 5% CO2. Cells were passaged every 3 days.

[0240] 16HBEge cells were grown in a medium containing MEM (Gibco, CAT# 11090099), 10% FBS, and penicillin and streptomycin (Gibco, CAT# 10378016) at 37 °C with 5% CO2. T75 flasks pre-coated with a coating solution containing LHC-8 basal medium (Gibco, CAT# 12677-027), bovine serum albumin 7.5% (Gibco, CAT# 15260-037), bovine collagen solution, Type 1 (Advanced BioMatrix, CAT# 5005), fibronectin from human plasma (ThermoFisher, CAT# 33016-015) were used for culturing 16HBEge cells. Cells were passaged every 4-5 days. Pre-coating was required for culturing cells in 96-well plates.RNP assembly

[0241] For cell culture experiments, RNPs were assembled at a 1.2:1 mole ratio of sgRNA (IDT or in vitro transcribed) to Cas protein in a supplier-recommended buffer (for nucleofection) or a phosphate buffer (25 mM NaPi, 150 mM NaCI, and 200 mM trehalose at pH 7.50) immediately before use; it is crucial to slowly add the Cas protein solution to the sgRNA solution while swirling (the reverse addition order can cause RNP aggregate formation). The solution was incubated for 15-25 min at room temperature or 5-10 min at 37 °C. For nucleofection, RNPs were further complexed with Alt-R Cas9 electroporation enhancer (100-nt ssDNA, IDT, CAT# 10007805) with a 1 :1 mole ratio of enhancer to RNP in supplier-recommended buffers (Lonza). For LNP assembly, RNPs (± ssDNA) were further diluted with a neutral solution of PBS / water (1 :1 , pH 7.3-7.5) or an acidic solution of sodium citrate (10 mM, pH 5.0) to a certain RNP concentration.Genome editing with different cell lines

[0242] Nucleofection: 250k NPCs or 200k HEK293T cells were nucleofected with 100 pmol pre-assembled RNP (with 100 pmol ssDNA enhancer) with program codes of EH- 100 and CM-130, respectively, according to the manufacturer’s instructions. Lonza SF (for HEK293T cells) and P3 (for tdTomato NPCs) buffers were used for the preparation of nucleofection mixtures (with a total volume of 20 pl). 10% of the nucleofected cells were transferred to 96-well plates. The culture media for NPCs was refreshed after 3 days; HEK293T cells were split with a ratio of 5:1 after 3 days. Cells were harvested for analysis after further incubation at 37°C for 2 days.

[0243] LNP delivery: 4-6.5k cells / well were seeded in 96-well plates 48 hours prior to LNP treatment (HEK293 cells: 4-5k, NPCs: 5-6k, and 16HBEge cells: 6-6.5k). The culture media was refreshed 24 hours after LNP treatment. HEK293T cells were split after 2additional days with a ratio of 1 :1 to 2:1 based on cell confluency. Cells were harvested for analysis after a total incubation time of 4-5 days (upon signal maturation for tdTom NPCs and HEK293 EGFP cells).

[0244] Cell viability was determined based on the counts of live cells (stained with trypan blue) at certain times post-treatment with LNPs, in comparison with cells treated with PBS (negative control).Flow cytometry

[0245] Cell fluorescence was assayed on an Attune NxT acoustic focusing cytometer (Thermo Fisher Scientific) equipped with a 554 nm excitation laser and 585 / 16 emission filter (tdTomato), 488 nm excitation laser and 530 / 30 emission filter (EGFP), and 400 nm excitation laser and 440 / 50 emission filter (BFP), and corresponding setup for cell-type analysis based on the antibody fluorophores. Data were analyzed using Attune Cytometric Software v5.1.1.LNP assembly and delivery experiment setup

[0246] LNP solutions with a total volume of less than 200 pL were prepared by pipet mixing; LNP solutions with higher volumes were prepared using a microfluidic mixing device, NANOGENERATOR Flex-M (PreciGenome).

[0247] Preparation of standard and cationic LNPs at small scales: RNPs were assembled by mixing iGeoCasO and sgRNAs at a mole ratio of 1 :1.2 and incubated for 20-30 min at room temperature. For HDR experiments, the assembled RNPs were further mixed with ssDNA templates at a mole ratio of 1:1 (ssDNA to RNP). The RNP stock solutions were diluted with an aqueous solution (PBS / water 1:1 , with 5 mM DTT, pH 7.3-7.5) to give a final RNP concentration of 5.0 or 7.5 pM. The lipid stock solutions in EtOH / DMSO were prepared at a total lipid concentration of 10-12 mg / mL. LNPs were assembled by pipet mixing with a volume ratio of 4:1 (aqueous to organic) and incubated at room temperature for 1 hour before being diluted with PBS (3X volume of the LNP solution) to give an LNP stock solution with RNP concentrations of 1.0 or 1.5 pM. For cell culture experiments, the LNP solutions were diluted with the corresponding culture media (9X volume of the LNP solution in PBS) and then used to treat cultured cells (in 1:1 volume ratio) with a final RNP cone, of 50 or 75 nM RNP (e.g., 5.0 or 7.5 pmol RNP in 100 uL culture media).

[0248] For long-term storage of standard and cationic LNPs at 4 °C, DTT was excluded during LNP assembly. A solution of DTT (5 mM) in PBS was used to activate LNPs right before the in vitro delivery experiments.

[0249] Preparation of FX12 and FC8 LNPs at small scales: RNPs were assembled by mixing iGeoCas9 and sgRNAs at a mole ratio of 1 :1.2, incubated for 10 min at 37 °C, and then complexed with 100-nt enhDNA (1.5 equiv. to RNP), and incubated for another 10 min at 37 °C, giving a stock solution of RNP with a concentration of 12-15 pM in the storage phosphate-trehalose buffer. The RNP stock solution was then diluted with sodium citrate buffer (10 mM, pH 5.0) to give a final RNP cone, of 1.06 pM (final pH ~5.2). The lipid stock solutions in EtOH / DMSO were prepared at a total lipid concentration of 10 mg / mL. LNPs were assembled by pipet mixing with a volume ratio of 4:1 (aqueous to organic) and incubated at room temperature for 20-30 minutes before being neutralized and diluted by PBS (3.24X volume of the LNP solution) to give an LNP stock solution with an RNP cone, of 0.2 pM. The LNP stock solution could be further diluted to give certain doses used for in vitro RNP delivery experiments. FC8 LNPs were DTT-activated during PBS dilution. For cell culture experiments, the LNP solutions were diluted with the corresponding culture media (9X volume of the LNP solution in PBS) and then used to treat cultured cells. For instance, 5 uL of the LNP stock solution (with RNP cone, of 0.2 pM) was diluted with 45 uL of culture media and used to treat mammalian cells in 50 uL culture media in a 96-well plate, giving a final RNP cone, of 10 nM (1 .0 pmol RNP in 100 uL culture media).

[0250] Microfluidic preparation of LNPs: RNPs were assembled by mixing iGeoCas9 and sgRNAs with a mole ratio of 1 :1.2, incubated for 10 min at 37 °C, and then complexed with 100-nt enhDNA (1.5 equiv. to RNP), and incubated for another 10 min at 37 °C, giving a stock solution of RNP with a concentration of 12-15 pM in the storage phosphate-trehalose buffer. The RNP stock solution was then diluted with sodium citrate buffer (10 mM, pH 5.0) to give a final RNP cone, of 1.25 pM (final pH ~5.2) or 0.625 pM (final pH ~5.1). The lipid stock solutions in EtOH / DMSO were prepared with a total lipid concentration of 10 mg / mL (for FX12m formulation) and 5 mg / mL (for FX8m formulation). FX12m LNPs were microfluidic-assembled with a volume ratio of 4:1 (aqueous to organic) at a flow rate of 3 mL / min; FC8m LNPs were microfluidic- assembled with a volume ratio of 4:1 (aqueous to organic) at a flow rate of 2 mL / min. The assembled LNPs were incubated at room temperature for 20-30 minutes before being subjected to dialysis against PBS using a dialysis membrane with a molecular weight cut-off of 10 kDa (ThermoFisher) overnight at 4 °C. Upon dialysis, the LNPs were concentrated by ultrafiltration using Amicon Ultra Centrifugal Filter with a molecular weight cut-off of 100 kDa (Millipore). FC8m LNPs were DTT-activated during the concentration step. The filter was washed three times with PBS to collectthe remaining LNPs absorbed on the filter membrane. The combined LNP solution was diluted with PBS to a certain volume used for animal experiments.Dynamic light scattering (DLS) assay

[0251] The size distribution of RNP particles or lipid nanoparticles was measured using Zetasizer (version 7.13, Malvern Panalytical; He-Ne Laser, A = 632 nm; detection angle = 173°).RNP encapsulation rate measurement

[0252] QUANT-IT RiboGreen RNA reagent (CAT# R11491) was used to estimate RNP encapsulation efficiency. LNP and lysed LNP (using 1% Triton X-100) samples were diluted using TE buffer to an estimated total nucleic acid concentration of 0.5-2.0 ng / uL. The diluted (lysed) LNP samples were mixed with the RiboGreen reagent (1 :1000 dilution in TE buffer) in a volume ratio of 1:1 (100 uL + 100 uL) and incubated at room temperature in the dark for 2 minutes before fluorescent signal measurement with the emission wavelength of 500 / 525 nm. The unencapsulated RNP proportion was estimated as the ratio of the signal intensity (with blank signal subtracted) of intact LNP to lysed LNP samples, thus giving the corresponding RNP encapsulation rate.Cryo-TEM image acquisition and processing

[0253] For cryo-TEM imaging, 3 pL of lipid nanoparticle suspension was added to a glow- discharged R 2 / 2 QUANTIFOIL Cu Grid (Ted Pella, Inc., Redding, CA). Samples were incubated for 20 seconds and blotted for 4 seconds (blotting force = 5) in a 4 °C high humidity chamber. After incubation, the samples were immediately plunged-frozen using an FEI Mark IV itrobot (FEI, Hillsboro, OR), resulting in vitreous ice. The samples were then imaged with an FEI Talos Arctica at 200 kV under low-dose conditions using a bottom-mount K3 camera (Gatan, Inc., Pleasanton, CA) at 36,000x magnification (0.5705 A / pixel). Images were analyzed with Cryo-SPARC software, and representative images were selected from multiple viewfields and grids.In vivo genome editing

[0254] Retro-orbital injections of LNPs consisting of different lipid formulations were performed with Ai9 tdTomato mice (C56BL / 6J, Jackson Laboratory, Bar Harbor, ME) and wild-type mice (BALB / c, Jackson Laboratory, Bar Harbor, ME), 10-16 weeks old, weighing 18-20 grams (male or female). The mice were sacrificed, and all tissues were collected for further analysis 2 weeks (Ai9 mice) or 10 days (wild-type mice) after LNP injection.

[0255] For flow analysis, isolated tissues were minced using a sterile blade and then subjected to digestion with collagenase type-1 (0.1 mg / mL as the final concentration, Gibco, CAT# 17018029) in 1 mL HBSS buffer (Gibco, CAT# 14175095) supplemented with 5 mM Ca2+at 37 °C for 2 hours with gentle shaking. Next, the digested solution was filtered using a 70-pm filter and quenched with PBS containing 2% FBS. A cell pellet was obtained by centrifuging for 5 min at a speed of 1500 xg at 4 °C. The supernatant was removed, and the cell pellet was resuspended in 1 ml of PBS containing 2% FBS, which could be used for flow analysis.

[0256] For cell type analysis, the dissociated tissue cells (100 pL) were incubated with corresponding antibodies (1 :200 dilution) for 1 hour in the dark at 4 °C. The stained cells were washed three times with 500 pL PBS and then resuspended in 500 pL PBS for flow cytometry analysis. The antibodies used for liver cell types were Alexa Fluor 647 anti-mouse CD95 (Fas) (BioLegend, CAT# 152620, for hepatocytes), Alexa Fluor 647 anti-mouse F4 / 80 (BioLegend, CAT# 157314, for macrophages), and Alexa Fluor 488 anti-mouse CD31 (BioLegend, CAT# 102414, for endothelial cells); the antibodies used for lung cell types were Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (BioLegend, CAT# 118212, for epithelial cells), Alexa Fluor 488 anti-mouse CD31 (BioLegend, CAT# 102414, for endothelial cells), and Pacific Blue anti-mouse CD45 (BioLegend, CAT# 157212, for immune cells).

[0257] For analysis by imaging, tissue blocks were embedded into optimal cutting temperature compounds (Sakura Finetek) and co-sectioned (8 pm) on a Cryostat instrument (Leica Biosystems) to prepare tissue sections. The mounted tissue slices were stained with DAPI before microscopy imaging. Images of tissue slices were taken using the Leica DMi8 microscope and analyzed using the Leica Application Suite X program (v 3.9.1.28433).

[0258] For analysis by NGS or DNA gel assays, dissociated tissue cells were collected and treated with Quick Extraction solution (Epicentre, Madison, Wl) to lyse the cells (65 °C for 20 min and then 95 °C for 20 min). The cell lysates were directly used for gene amplicon prep by PCR.Next-generation sequencing

[0259] Edited cells were harvested and treated with Quick Extraction solution (Epicentre, Madison, Wl) to lyse the cells (65 °C for 20 min and then 95 °C for 20 min). The cell lysates were directly used for gene amplicon prep by PCR. Amplicons of genomic targets were PCR-amplified in the presence of corresponding primers, which were designed to have no overlap with their corresponding donor ssDNA sequence in thecase of HDR. The PCR products were purified with magnetic beads (Berkeley Sequencing Core Facility) before being subjected to next-generation sequencing (NGS) with MiSeq (Illumina) at 2x300 bp with a depth of at least 20,000 reads per sample. The sequencing reads were subjected to CRISPResso2 (https: / / github.com / pinellolab / CRISPResso2) to quantify the levels of indels and HDR. Immunogenicity assessment

[0260] Wild-type BALB / c mice (male or female), 10-16 weeks old, weighing 18-20 grams, were utilized for cytokine measurement experiments. LNP complexes based on FX12m or FC8m formulations with RNP cargo or as empty vectors, RNP-only solution, PBS (negative control), and LPS (lipopolysaccharide, with a dose of 1 mg / kg; positive control) were administered via the retro-orbital route (intravenous). LNPs (with or without RNPs) were injected at doses following the experimental setup (e.g., 4.6 mg / kg RNP for FX12m formulation). Injections were performed with a total volume of 150 pL per mouse.

[0261] At time points of 6 and 24 hours, the first two batches of mice were humanely euthanized, and corresponding blood samples were collected in heparin and centrifuged at 1500 xg for 10 minutes at 4 °C. The levels of cytokines, including interleukin 2 (IL-2, ELISA kit from R&D Systems, CAT# DY402-05), interleukin 6 (IL-6, ELISA kit from R&D Systems, CAT# DY406-05), macrophage inflammatory protein 2 (MIP-2, ELISA kit from R&D Systems, CAT# DY452-05), and tumor necrosis factor a (TNF-a, ELISA kit from R&D Systems, CAT# DY410-05) in the plasma were determined based on ELISA assays following the manufacturer’s protocols (R&D Systems).

[0262] Another batch of mice were humanely euthanized two weeks post-injection, and blood samples were collected in heparin and centrifuged at 1500 xg for 10 minutes at 4 °C. The levels of liver damage enzymes, including alanine aminotransferase (ALT ELISA kit from Abeam, CAT# ab282882), aspartate aminotransferase (AST ELISA kit from Abeam, CAT# ab263882), and transglutaminase 2 (TGM2 ELISA kit from RayBiotech, CAT# ELM-TGM2-1) in the plasma were determined based on ELISA assays following manufacturers’ protocols (Abeam or RayBiotech).

[0263] References1Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. & Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).2Cong, L, Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. & Zhang, F. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013).3Wang, J. Y. & Doudna, J. A. CRISPR technology: A decade of genome editing is only the beginning. Science 379, eadd8643 (2023).4Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR-Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490-507 (2019).5Katti, A., Diaz, B. J., Caragine, C. M., Sanjana, N. E. & Dow, L. E. CRISPR in cancer biology and therapy. Nat. Rev. Cancer 22, 259-279 (2022).6Sharma, G., Sharma, A. R., Bhattacharya, M., Lee, S., & Chakraborty, C. CRISPR- Cas9: A preclinical and clinical perspective for the treatment of human diseases. Mol. Ther. 29, 571-586 (2021).7van Haasteren, J., Li, J., Scheideler, O. J., Murthy, N. & Schaffer, D. V. The delivery challenge: fulfilling the promise of therapeutic genome editing. Nat. Biotech. 38, 845- 855 (2020).8Lino, C. A., Harper, J. C., Carney, J. P. & Timlin, J. A. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 25, 1234-1257 (2018).9Xu, C. L., C. Ruan, M. Z., Mahajan, V. B., & Tsang, S. H. Viral delivery systems for CRISPR. Viruses 11 , 28 (2019).10Khirallah, J., Eimbinder, M., Li, Y., & Xu, Q. Clinical progress in genome-editing technology and in vivo delivery techniques. Trends in Genetics 39, 208-216 (2023).11Zhao, Z., Anselmo, A. C., & Mitragotri, S. Viral vector-based gene therapies in the clinic. Bioeng. Transl. Med. 7, e 10258 (2022).12Lan, T., Que, H., Luo, M., Zhao, X., Wei, X. Genome editing via non-viral delivery platforms: current progress in personalized cancer therapy. Mol. Cancer 21 , 71 (2022).13Kazemian, P., Yu, S.-Y., Thomson, S. B., Birkenshaw, A., Leavitt, B. R. & Ross, C. J. D. Lipid-nanoparticle-based delivery of CRISPR / Cas9 genome-editing components. Mol. Pharm. 19, 1669-1686 (2022).14Rothgangl, T., Dennis, M. K., Lin, P. J., Oka, R., Witzigmann, D., Villiger, L, Qi, W., Hruzova, M., Kissling, L., Lenggenhager, D., Borrelli, C., Egli, S., Frey, N., Bakker, N.,Walker, J. A., Kadina, A. P., Victorov, D. V., Pacesa, M., Kreutzer, S., Kontarakis, Z., Moor, A., Jinek, M., Weissman, D., Stoffel, M., Boxtel, R., Holden, K., Pardi, N., Thbny, B., Haberle, J., Tam, Y. K., Semple, S. C. & Schwank, G. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat. Biotech. 39, 949- 957 (2021).15Musunuru, K., Chadwick, A. C., Mizoguchi, T., Garcia, S. P., DeNizio, J. E., Reiss,C. W., Wang, K., Iyer, S., Dutta, C., Clendaniel, V., Amaonye, M., Beach, A., Berth, K., Biswas, S., Braun, M. C., Chen, H., Colace, T. V., Ganey, J. D., Gangopadhyay, S. A., Garrity, R., Kasiewicz, L. N., Lavoie, J., Madsen, J. A., Matsumoto, Y., Mazzola, A.M., Nasrullah, Y. S., Nneji, J., Ren, H., Sanjeev, A., Shay, M., Stahley, M. R., Fan, S.H. Y., Tam, Y. K., Gaudelli, N. M., Ciaramella, G., Stolz, L. E., Malyala, P., Cheng, C. J., Rajeev, K. G., Rohde, E., Bellinger, A. M. & Kathiresan, S. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature, 593, 429-434 (2021).16Breda, L, Papp, T. E., Triebwasser, M. P., Yadegari, A., Fedorky, M. T., Tanaka,N., Abdulmalik, O., Pavani, G., Wang, Y., Grupp, S. A., Chou, S. T., Ni, H., Mui, B. L, Tam, Y. K., Weissman, D., Rivella, S., & Parhiz, H. In vivo hematopoietic stem cell modification by mRNA delivery. Science 381 , 436-443 (2023).17Cheng, Q., Wei, T., Farbiak, L., Johnson, L. T., Dilliard, S. A. & Siegwart, D. J. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat. Nanotech. 15, 313-320 (2020).18Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld,I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., Bacchetta, R., Tsalenko, A., Dellinger,D., Bruhn, L. & Porteus, M. H. Chemically modified guide RNAs enhance CRISPR- Cas genome editing in human primary cells. Nat. Biotech. 33, 985-989 (2015).19Hou, X., Zaks, T., Langer, R., & Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6, 1078-1094 (2021).20Zhang, S., Shen, J., Li, D., & Cheng, Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics, 11 , 614-648 (2021).21Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L, Hu, J. H., Maeder, M. L., Joung, J. K., Chen, Z., & Liu, D. R. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat.Biotech. 33, 73-80 (2015).22Sung, Y. H., Kim, J. M., Kim, H.-T., Lee, J., Jeon, J., Jin, Y., Choi, J.-H., Ban, Y. H., Ha, S.-J., Kim, C.-H., Lee, H.-W. & Kim, J.-S. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 24, 125-131 (2014).23Zou, R. S., Liu, Y., Wu, B., Ha, T. Cas9 deactivation with photocleavable guide RNAs. Mol. Ce / / 81, 1553-1565 (2021).24Huang, C.; Han, Z.; Evangelopoulos, M. & Mirkin, C. A. CRISPR spherical nucleic acids. J. Am. Chem. Soc. 144, 18756-18760 (2022).25Sun, W., Ji, W., Hall, J. M., Hu, Q., Wang, C., Beisel, C. L. & Gu, Z. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. 54, 12029-12033 (2015).26Wang, Y., Wang, X., Xie, R., Burger, J. C., Tong, Y., & Gong, S. Overcoming the blood-brain barrier for gene therapy via systemic administration of GSH-responsive silica nanocapsules. Adv. Mater. 35, 2208018 (2023).27Alsaiari, S. K.; Patil, S.; Alyami, M.; Alamoudi, K. O.; Aleisa, F. A.; Merzaban, J. S.; Li, M. & Khashab, N. M. Endosomal escape and delivery of CRISPR / Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. J. Am. Chem. Soc. 140, 143-146 (2018).28Wang, M., Zuris, J. A., Meng, F., Rees, H., Sun, S., Deng, P., Han, Y., Gao, X., Pouli, D., Wu, Q., Georgakoudi, I., Liu, D. R. & Xu, Q. Efficient delivery of genomeediting proteins using bioreducible lipid nanoparticles. Proc. Natl Acad. Sci. USA 113, 2868-2873 (2016).29Wei, T., Cheng, Q., Min, Y., Olson, E. N. & Siegwart, D. J. Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing. Nat. Comm. 11, 3232 (2020).30Onuma, H., Sato, Y. & Harashima, H. Lipid nanoparticle-based ribonucleoprotein delivery for in vivo genome editing. J. Control. Release 355, 406-416 (2023).31Lee, B., Lee, K., Panda, S., Chong, A., Bugay, V., Park, H. M., Brenner, R., Murthy, N., & Lee, H. Y. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat. Biomed. Eng. 2, 497-507 (2018).32Lee, K., Conboy, M., Park, H. M., Jiang, F., Kim, H. J., Dewitt, M. A., Mackley, V. A., Chang, K., Rao, A., Skinner, C., Shobha, T., Mehdipour, M., Liu, H., Huang, W., Lan, F., Bray, N. L, Li, S., Corn, J. E., Kataoka, K., Doudna, J. A., Conboy, I. & Murthy, N. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889-901 (2017).33Staahl, B. T., Benekareddy, M., Coulon-Bainier, C., Banfal, A. A., Floor, S. N., Sabo, J. K., Urnes, C., Munares, G. A., Ghosh, A. & Doudna, J. A. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat. Biotech. 35, 431-434 (2017).34Chen, K., Stahl, E. C., Kang, M. H., Xu, B., Allen, R., Trinidad, M., & Doudna, J. A. Engineering self-deliverable ribonucleoproteins for genome editing in the brain. Nat. Commun. 15, 1727 (2024).35Jung, H. N., Lee, Y., Lee, S., Youn, H., & Im, J. Lipid nanoparticles for delivery of RNA therapeutics: Current status and the role of in vivo imaging. Theranostics 12, 7509-7531 (2022).36Harrington, L. B., Paez-Espino, D., Staahl, B. T., Chen, J. S., Ma, E., Kyrpides, N. C. & Doudna, J. A. A thermostable Cas9 with increased lifetime in human plasma. Nat. Commun. 8, 1424 (2017).37Mougiakos, L, Mohanraju, P., Bosma, E. F., Vrouwe, V., Finger Bou, M., Naduthodi, M. I., Gussak, A., Brinkman, R. B. & van Kranenburg, R. Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Nat. Comm. 8, 1647 (2017).38Katzmann, J. L., Cupido, A. J., & Laufs, U. Gene therapy targeting PCSK9. Metabolites 12, 70 (2022).39Li, B., Manan, R. S., Liang, S., Gordon, A., Jiang, A., Varley, A., Gao, G., Langer, R., Xue, W., & Anderson, D. Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing. Nat. Biotech. 41 , 1410-1415 (2023).40Qiu, M., Tang, Y., Chen, J., Muriph, R., Ye, Z., Huang, C., Evans, J., Henske, E. P., & Xu, Q. Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis. Proc. Natl. Acad. Sci. U.S.A. 119, e2116271119 (2022).41Liang, S., Walkey, C. J., Martinez, A. E., Su, Q., Dickinson, M. E., Wang, D., Lagor,W. R., Heaney, J. D., Gao, G., & Xue, W. AAV5 delivery of CRISPR-Cas9 supports effective genome editing in mouse lung airway. Mol. Ther. 30, 238-243 (2022).42Sun, Y., Chatterjee, S., Lian, X., Traylor, Z., Sattiraju, S. R., Xiao, Y., Dilliard, S. A., Sung, C., Kim, M., Lee, S. M., Moore, S., Wang, X., Zhang, D., Wu, S., Basak, P., Wang, J., Liu, J., Mann, R. J., LePage, D. F., Jiang, W., Abid, S., Hennig, M., Martinez, A., Wustman, B. A., Lockhart, D. J., Jain, R., Conlon, R. A., Drumm, M. L., Hodges, C. A., Siegwart, D. J. In vivo editing of lung stem cells for durable gene correction in mice. Science 384, 1196-1202 (2024).43Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., Gonzales, A. P., Li, Z., Peterson, R. T., Yeh, J., Aryee, M. J. & Joung, J. K. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481-485 (2015).44Zhang, L., Zuris, J. A., Viswanathan, R., Edelstein, J. N., Turk, R., Thommandru, B., Rube, H. T., Glenn, S. E., Collingwood, M. A., Bode, N. M., Beaudoin, S. F., Lele, S., Scott, S. N., Wasko, K. M., Sexton, S., Borges, C. M., Schubert, M. S., Kurgan, G. L, McNeill, M. S., Fernandez, C. A., Myer, V. E., Morgan, R. A., Behlke, M. A. & Vakulskas, C. A. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat. Comm. 12, 3908 (2021).45Ma, E„ Chen, K., Shi, H„ Stahl, E. C„ Adler, B„ Trinidad, M„ Liu, J., Zhou, K„ Ye, J. & Doudna, J. A. Improved genome editing by an engineered CRISPR-Cas12a. Nucleic Acids Res. 50, 12689-12701 (2022).46Eggers, A. R., Chen, K., Soczek, K. M., Tuck, K. T., Doherty, E. E., Xu, B., Trinidad, M. I., Thornton, B. W., Yoon, P. H., Doudna, J. A. Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9. Cell 187, 3249-3261 (2024).47Zhao, S., Gao, K., Han, H., Stenzel, M., Yin, B., Song, H., Sharma, R., Arogundade, O. H., Paul, A., Tuma, J., Collins, M. G., Burgstone, B. W., Perez, B. S., Chen, Y.-J., Smith, A., Lee, H., Wang, A., Murthy, N. Acid-degradable lipid nanoparticles enhance the delivery of mRNA. Nat. Nanotech. (2024). DOI: 10.1038 / S41565-024-01765-4.48Mitchell, M. J., Billingsley, M. M., Haley, R. M., Wechsler, M. E., Peppas, N. A. & Langer, R. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discovery 20, 101-124 (2021).49Suk, J. S., Xu, Q., Kim, N., Hanes, J. & Ensign, L. M. PEGylation as a strategy forimproving nanoparticle-based drug and gene delivery. Adv. Drug Deliv. Rev. 99, 28- 51 (2016).50Haseda, Y., Munakata, L., Meng, J., Suzuki, R., & Aoshi, T. Microfluidic-prepared DOTAP nanoparticles induce strong T-cell responses in mice. PLoS ONE 15, e0227891 (2020).51Finn, J. D., Smith, A. R., Patel, M. C., Shaw, L, Youniss, M. R., Van Heteren, J., Dirstine, T., Ciullo, C., Lescarbeau, R., Seitzer, J., Shah, R. R., Shah, A., Ling, D., Growe, J., Pink, M., Rohde, E., Wood, K. M., Salomon, W. E., Harrington, W. F., Dombrowski, C., Strapps, W. R., Chang, Y., Morrissey, D. V. A single administration of CRISPR / Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227-2235 (2018).52Gautam, M., Jozic, A., Su, G. L., Curtis, A., Arrizabalaga, S., Tschetter, W., Ryals, R. C., & Sahay, G. Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina. Nat. Commun. 14, 6468 (2023).53Alapati, D., Zacharias, W. J., Hartman, H. A., Rossidis, A. C., Stratigis, J. D., Ahn, N. J., Coons, B., Zhou, S., Li, H., Singh, K., Katzen, J., Tomer, Y., Chadwick, A. C., Musunuru, K., Beers, M. F., Morrisey, E. E., & Peranteau, W. H. In utero gene editing for monogenic lung disease. Sci. Transl. Med. 11 , eaav8375 (2019).54Tong, S.; Moyo, B.; Lee, C. M.; Leong, K.; Bao, G. Engineered materials for in vivo delivery of genome-editing machinery. Nat. Rev. Mater. 4, 726-737 (2019).55Bloom, J. D., Labthavikul, S. T., Otey, C. R. & Arnold, F. H. Protein stability promotes evolvability. Proc. Natl. Acad. Sci. U.S.A. 103, 5869-5874 (2006).56Eygeris, Y.; Gupta, M.; Kim, J. & Sahay, G. Chemistry of lipid nanoparticles for RNA delivery. Acc. Chem. Res. 55, 2-12 (2022).57Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotech. 38, 824-844 (2020).58Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A. & Liu, D. R. Search-and- replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019).59Rees, H. A., & Liu, D. R. Base editing: Precision chemistry on the genome andtranscriptome of living cells. Nat. Rev. Genet. 19, 770-788 (2018).60Dilliard, S. A., Cheng, Q. & Siegwart, D. J. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 118, e2109256118 (2021).

[0264] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0265] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. , any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0266] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

1. CLAIMSWhat is claimed is:

1. A lipid nanoparticle (LNP) formulated for delivery of a payload to a target cell, comprising:(a) an ionizable lipid present in a mole percentage of about 36-56% of the total lipids;(b) a neutral phospholipid present in a mole percentage of about 8-20% of the total lipids;(c) cholesterol present in a mole percentage of about 30-50% of the total lipids;(d) a pegylated lipid present in a mole percentage of about 1-4% of the total lipids;(e) a CRISPR-Cas effector protein or an mRNA encoding the CRISPR-Cas effector protein; and(f) a guide RNA.

2. The LNP of claim 1 , wherein the ionizable lipid is present in a mole percentage of about 40- 52% of the total lipids.

3. The LNP of claim 1 , wherein the ionizable lipid is present in a mole percentage of about 46% of the total lipids.

4. The LNP of any one of claims 1-3, wherein the ionizable lipid is BP lipid 312 or LP01 .

5. The LNP of any one of claims 1-4, wherein the neutral phospholipid is present in a mole percentage of about 10-15% of the total lipids.

6. The LNP of any one of claims 1-5, wherein the neutral phospholipid is DOPE.

7. The LNP of any one of claims 1-6, wherein the cholesterol is present in a mole percentage of about 35-45% of the total lipids.

8. The LNP of any one of claims 1-6, wherein the cholesterol is present in a mole percentage of about 40% of the total lipids.

9. The LNP of any one of claims 1-8, wherein the pegylated lipid is present in a mole percentage of about 1.5% of the total lipids.

10. The LNP of any one of claims 1-8, wherein the pegylated lipid is present in a mole percentage of about 1.2% of the total lipids.

11. The LNP of any one of claims 1-10, wherein the pegylated lipid is DMG-PEG-2000.

12. The LNP of any one of claims 1-11 , further comprising a pegylated cholesterol.

13. The LNP of claim 12, wherein the pegylated cholesterol is present in a mole percentage of about 0.2-1% of the total lipids.

14. The LNP of claim 12, wherein the pegylated cholesterol is present in a mole percentage of about 0.4% of the total lipids.

15. The LNP of any one of claims 1-14, wherein the pegylated cholesterol is Chol-PEG-2000.

16. A lipid nanoparticle (LNP) formulated for delivery of a payload to a target cell, comprising:(a) a cationic lipid present in a mole percentage of about 30-55% of the total lipids;(b) an ionizable lipid present in a mole percentage of about 15-35% of the total lipids;(c) a neutral phospholipid present in a mole percentage of about 8-20% of the total lipids;(d) cholesterol present in a mole percentage of about 10-25% of the total lipids;(e) a pegylated lipid present in a mole percentage of about 0.8-4% of the total lipids;(f) a CRISPR-Cas effector protein or an mRNA encoding the CRISPR-Cas effector protein; and(g) a guide RNA.

17. The LNP of claim 16, wherein the cationic lipid is present in a mole percentage of about 40- 48% of the total lipids.

18. The LNP of claim 16, wherein the cationic lipid is present in a mole percentage of about 42% of the total lipids.

19. The LNP of claim 16, wherein the cationic lipid is present in a mole percentage of about 46% of the total lipids.

20. The LNP of any one of claims 16-19, wherein the cationic lipid is ADC.

21. The LNP of any one of claims 16-20, wherein the ionizable lipid is present in a mole percentage of about 20-30% of the total lipids.

22. The LNP of any one of claims 16-20, wherein the ionizable lipid is present in a mole percentage of about 24% of the total lipids.

23. The LNP of any one of claims 16-20, wherein the ionizable lipid is present in a mole percentage of about 26% of the total lipids.

24. The LNP of any one of claims 16-23, wherein the ionizable lipid is Lipid III-45 or ALC-0315.

25. The LNP of any one of claims 16-24, wherein the neutral phospholipid is present in a mole percentage of about 11-16% of the total lipids.

26. The LNP of any one of claims 16-24, wherein the neutral phospholipid is present in a mole percentage of about 12% of the total lipids.

27. The LNP of any one of claims 16-24, wherein the neutral phospholipid is present in a mole percentage of about 15% of the total lipids.

28. The LNP of any one of claims 16-27, wherein the neutral phospholipid is DOPE.

29. The LNP of any one of claims 16-28, wherein the cholesterol is present in a mole percentage of about 14-20% of the total lipids.

30. The LNP of any one of claims 16-28, wherein the cholesterol is present in a mole percentage of about 16% of the total lipids.

31. The LNP of any one of claims 16-30, wherein the pegylated lipid is present in a mole percentage of about 1-3% of the total lipids.

32. The LNP of any one of claims 16-30, wherein the pegylated lipid is present in a mole percentage of about 1.5% of the total lipids.

33. The LNP of any one of claims 16-32, wherein the pegylated lipid is DMG-PEG-2000.

34. The LNP of any one of claims 1-33, wherein the LNP comprises the CRISPR-Cas effector protein complexed with a guide RNA as a ribonucleoprotein (RNP).

35. The LNP of any one of claims 1-34, wherein the CRISPR-Cas effector protein is a Cas9 protein.

36. The LNP of any one of claims 1-34, wherein the CRISPR-Cas effector protein is a Cas9 protein that includes an amino acid sequence that is 80% or more identical to the iGeoCas9 sequence of SEQ ID NO: 34.

37. A method of making the LNP of any one of claims 1-36, the method comprising:(a) combining the CRISPR-Cas effector protein with a guide RNA, thereby forming an ribonucleoprotein (RNP) mixture; or combining the mRNA encoding the CRISPR-Cas effector protein with the guide RNA, thereby forming a nucleic acid mixture;(b) combining the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid to form a lipid mixture; and(c) combining, at a pH of about 5, the lipid mixture with the RNP mixture, or the lipid mixture with the nucleic acid mixture; thereby producing the LNP.

38. The method of claim 37, wherein the combining of step (a) is performed in the presence of a single stranded DNA (ssDNA) enhancer DNA (enhDNA) or anionic polymer, (e.g., poly L- glutamate with a molecular weight of 15-50 kDa).

39. The method of claim 37 or claim 38, wherein, during the combining of step (b), the cationic lipid is combined with the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid to form the lipid mixture.

40. A method of delivering a molecular payload to a eukaryotic cell, the method comprising contacting the eukaryotic cell with the LNP of any one of claims 1-36, thereby providing the CRISPR-Cas effector protein and a guide RNA to the eukaryotic cell.41 . The method of claim 40, wherein the eukaryotic cell is a liver cell.

42. The method of claim 40, wherein the eukaryotic cell is a lung cell.

43. The method of claim 40, wherein the eukaryotic cell is a spleen, kidney, or heart cell.

44. The method of any one of claims 40-43, wherein the eukaryotic cell is in vitro or ex vivo.

45. The method of any one of claims 40-43, wherein the eukaryotic cell is in vivo.

46. The method of claim 40, wherein the eukaryotic cell is in vivo, wherein the LNP comprises the CRISPR-Cas effector protein complexed with the guide RNA as a ribonucleoprotein (RNP), and wherein said contacting comprises intravenous administration of the LNP to an individual.

47. The method of claim 46, wherein the method results in editing of DNA primarily in cells of the liver.

48. The method of claim 46, wherein the method results in editing of DNA primarily in cells of the lung.

Images