Gene editing therapy for cardiovascular diseases

Abstract

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA-targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD.

Keywords

cardiovascular disease / CRISPR/Cas / gene therapy / lipid nanoparticles

References

1	G Mendel. Experiments in plant hybridization. Meetings of the Natural Science Society. February/March 1965.
2	N Roll-Hansen. The crucial experiment of Wilhelm Johannsen. Biol Philos. 1989;4(3):303-329.
3	EH Szybalska, W Szybalski. Genetics of human cell lines, IV. DNA-mediated heritable transformation of a biochemical trait. P Natl Acad Sci USA. 1962;48(12):2026-2034.
4	RM Blaese, KW Culver, AD Miller, et al. T lymphocyte-directed gene therapy for ADA$^-$ SCID: initial trial results after 4 years. Science. 1995;270(5235):475-480.
5	X Song, C Liu, N Wang, et al. Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy. Adv Drug Deliv Rev. 2021;168:158-180.
6	A Karimian, N Gorjizadeh, F Alemi, et al. CRISPR/Cas9 novel therapeutic road for the treatment of neurodegenerative diseases. Life Sci. 2020;259:118165.
7	S Yla-Herttuala, AH Baker. Cardiovascular gene therapy: past, present, and future. Mol Ther. 2017;25(5):1095-1106.
8	EM Kennedy, BR Cullen. Gene editing: a new tool for viral disease. Annu Rev Med. 2017;68:401-411.
9	E Tikkanen, S Gustafsson, E Ingelsson. Associations of fitness, physical activity, strength, and genetic risk with cardiovascular disease: longitudinal analyses in the UK Biobank Study. Circulation (New York, NY). 2018;137(24):2583-2591.
10	GH Gibbons, CE Seidman, EJ Topol. Conquering atherosclerotic cardiovascular disease—50 years of progress. New Engl J Med. 2021;384(9):785-788.
11	FLJ Visseren, F MacH, YM Smulders, et al. 2021 ESC Guidelines on cardiovascular disease prevention in clinical practice. Eur Heart J. 2021;42(34):3227-3337.
12	O Smithies, RG Gregg, SS Boggs, MA Koralewski, RS Kucherlapati. Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature (London). 1985;317(6034):230-234.
13	KR Thomas, MR Capecchi. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51(3):503-512.
14	Y-G Kim, J Cha, S Chandrasegaran. Hybrid restriction enzymes: zinc finger fusions to Fok i cleavage domain. Proc Natl Acad Sci U S A. 1996;93(3):1156-1160.
15	M Bibikova, M Golic, KG Golic, D Carroll. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics (Austin). 2002;161(3):1169-1175.
16	J Wang, EJ Rebar, DY Guschin, et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007;25(7):778-785.
17	DJ Segal, J Büchel, M Szczepek, T Cathomen, L Serrano, V Brondani. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol. 2007;25(7):786-793.
18	MJ Moscou, AJ Bogdanove. Simple cipher governs DNA recognition by TAL effectors. Science. 2009;326(5959):1501.
19	J Boch, H Scholze, S Schornack, et al. Breaking the code of DNA binding specificity of TAL-Type III effectors. Science. 2009;326(5959):1509-1512.
20	T Sakuma, T Yamamoto. Updated overview of TALEN construction systems. Methods Mol Biol. 2023;2637:27-39.
21	SR Bacman, JHK Kauppila, CV Pereira, et al. MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation. Nat Med. 2018;24(11):1696-1700.
22	Y Ishino, H Shinagawa, K Makino, M Amemura, A Nakata. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987;169(12):5429-5433.
23	FJM Mojica, C Díez-Villase?or, E Soria, G Juez. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol. 2000;36(1):244-246.
24	R Jansen, JDAv Embden, W Gaastra, LM Schouls. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565-1575.
25	FJM Mojica, C Díez-Villase?or, J García-Martínez, E Soria. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005;60(2):174-182.
26	C Pourcel, G Salvignol, G Vergnaud. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology (Reading). 2005;151(3):653-663.
27	A Bolotin, B Quinquis, A Sorokin, S Dusko Ehrlich. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology (Reading). 2005;151(8):2551-2561.
28	KS Makarova, NV Grishin, SA Shabalina, YI Wolf, EV Koonin. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006;1(1):7-7.
29	R Barrangou, C Fremaux, H Deveau, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315(5819):1709-1712.
30	SJJ Brouns, MM Jore, M Lundgren, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008;321(5891):960-964.
31	LA Marraffini, EJ Sontheimer. CRISPR interference limits horizontal gene transfer in Staphylococci by targeting DNA. Science. 2008;322(5909):1843-1845.
32	FJM Mojica, C Diez-Villasenor, J Garcia-Martinez, C Almendros. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading). 2009;155(3):733-740.
33	S Moineau, JE Garneau, M-è Dupuis, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature (London). 2010;468(7320):67-71.
34	E Deltcheva, K Chylinski, CM Sharma, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature (London). 2011;471(7340):602-607.
35	M Jinek, K Chylinski, I Fonfara, M Hauer, JA Doudna, E Charpentier. A programmable dual-RNA—guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.
36	R Sapranauskas, G Gasiunas, C Fremaux, R Barrangou, P Horvath, V Siksnys. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011;39(21):9275-9282.
37	G Gasiunas, R Barrangou, P Horvath, V Siksnys. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012;109(39):E2579-E2586.
38	L Cong, FA Ran, D Cox, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819-823.
39	P Mali, L Yang, KM Esvelt, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826.
40	S Qi Lei, H Larson Matthew, A Gilbert Luke, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152(5):1173-1183.
41	D Bikard, W Jiang, P Samai, A Hochschild, F Zhang, LA Marraffini. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429-7437.
42	A Gilbert Luke, A Horlbeck Max, B Adamson, et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159(3):647-661.
43	H Wang, H Yang, CS Shivalila, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910-918.
44	G Schwank, B-K Koo, V Sasselli, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013;13(6):653-658.
45	Y Wu, D Liang, Y Wang, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell. 2013;13(6):659-662.
46	P Horvath, R Barrangou. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327(5962):167-170.
47	KS Makarova, YI Wolf, J Iranzo, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Micro. 2020;18(2):67-83.
48	TW Lo, CS Pickle, S Lin, et al. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics. 2013;195(2):331-348.
49	R Scully, A Panday, R Elango, NA Willis. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol. 2019;20(11):698-714.
50	N Kolli, M Lu, P Maiti, J Rossignol, GL Dunbar. CRISPR-Cas9 mediated gene-silencing of the mutant huntingtin gene in an in vitro model of huntington's disease. Int J Mol Sci. 2017;18(4):754.
51	AR Eggers, K Chen, KM Soczek, et al. Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9. Cell. 2024.
52	BP Kleinstiver, MS Prew, SQ Tsai, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015;523(7561):481-485.
53	JH Hu, SM Miller, MH Geurts, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018;556(7699):57-63.
54	H Nishimasu, X Shi, S Ishiguro, et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science. 2018;361(6408):1259-1262.
55	P Chatterjee, N Jakimo, JM Jacobson. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci Adv. 2018;4(10):eaau0766-eaau0766.
56	P Chatterjee, N Jakimo, J Lee, et al. An engineered ScCas9 with broad PAM range and high specificity and activity. Nat Biotechnol. 2020;38(10):1154-1158.
57	H Deveau, R Barrangou, JE Garneau, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol. 2008;190(4):1390-1400.
58	Z Hou, Y Zhang, NE Propson, et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA. 2013;110(39):15644-15649.
59	A Edraki, A Mir, R Ibraheim, et al. A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Mol Cell. 2019;73(4):714-726.
60	FA Ran, L Cong, WX Yan, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520(7546):186-191.
61	BP Kleinstiver, MS Prew, SQ Tsai, et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol. 2015;33(12):1293-1298.
62	D Ma, Z Xu, Z Zhang, et al. Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information. Nat Commun. 2019;10(1):560.
63	S Wang, H Mao, L Hou, et al. Compact SchCas9 recognizes the simple NNGR PAM. Adv Sci (Weinh). 2022;9(4):e2104789.
64	E Kim, T Koo, SW Park, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun. 2017;8:14500.
65	LB Harrington, D Paez-Espino, BT Staahl, et al. A thermostable Cas9 with increased lifetime in human plasma. Nat Commun. 2017;8(1):1424.
66	S Acharya, A Mishra, D Paul, et al. Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing. Proc Natl Acad Sci USA. 2019;116(42):20959-20968.
67	H Hirano, JS Gootenberg, T Horii, et al. Structure and engineering of Francisella novicida Cas9. Cell. 2016;164(5):950-961.
68	RT Walton, KA Christie, MN Whittaker, BP Kleinstiver. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science. 2020;368(6488):290-296.
69	TP Huang, ZJ Heins, SM Miller, et al. High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs. Nat Biotechnol. 2023;41(1):96-107.
70	VP Chauhan, PA Sharp, R Langer. Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases. Proc Natl Acad Sci U S A. 2023;120(11):e2300605120-e2300605120.
71	L Zhao, SRT Koseki, RA Silverstein, et al. PAM-flexible genome editing with an engineered chimeric Cas9. Nat Commun. 2023;14(1):6175-6175.
72	AC Komor, YB Kim, MS Packer, JA Zuris, DR Liu. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533(7603):420-424.
73	K Nishida, T Arazoe, N Yachie, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016;353(6305):1248-1248.
74	Y Ma, J Zhang, W Yin, Z Zhang, Y Song, X Chang. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat Methods. 2016;13(12):1029-1035.
75	GT Hess, L Frésard, K Han, et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat Methods. 2016;13(12):1036-1042.
76	NM Gaudelli, AC Komor, HA Rees, et al. Programmable base editing of A?T to G?C in genomic DNA without DNA cleavage. Nature (London). 2017;551(7681):464-471.
77	MJ Landrum, JM Lee, M Benson, et al. Public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 2016;44(1):D862-D868.
78	E Zhang, ME Neugebauer, NA Krasnow, DR Liu. Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference. Nat Commun. 2024;15(1):1697-1697.
79	X Zhang, B Zhu, L Chen, et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat Biotechnol. 2020;38(7):856-860.
80	J Grunewald, R Zhou, CA Lareau, et al. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nat Biotechnol. 2020;38(7):861-864.
81	RC Sakata, S Ishiguro, H Mori, et al. Base editors for simultaneous introduction of C-to-T and A-to-G mutations. Nat Biotechnol. 2020;38(7):865-869.
82	C Li, R Zhang, X Meng, et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat Biotechnol. 2020;38(7):875-882.
83	IC Kurt, R Zhou, S Iyer, et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol. 2021;39(1):41-46.
84	D Zhao, J Li, S Li, et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol. 2021;39(1):35-40.
85	H Tong, N Liu, Y Wei, et al. Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase. Natl Sci Rev. 2023;
86	H Tong, X Wang, Y Liu, et al. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nat Biotechnol.
87	L Chen, M Hong, C Luan, et al. Adenine transversion editors enable precise, efficient A*T-to-C*G base editing in mammalian cells and embryos. Nat Biotechnol. 2023.
88	L Ye, D Zhao, J Li, et al. Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells. Nat Biotechnol. 2024.
89	YB Kim, AC Komor, JM Levy, MS Packer, KT Zhao, DR Liu. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol. 2017;35(4):371-376.
90	J Grünewald, R Zhou, SP Garcia, et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature (London). 2019;569(7756):433-437.
91	MP Zafra, EM Schatoff, A Katti, et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat Biotechnol. 2018;36(9):888-896.
92	JM Gehrke, O Cervantes, MK Clement, et al. An apobec3a-cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol. 2018;36(10):977-982.
93	X Wang, J Li, Y Wang, et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat Biotechnol.
94	Z Liu, S Chen, H Shan, et al. Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions. BMC Biol. 2020;18(1):111-111.
95	W Yu, J Li, S Huang, et al. Harnessing A3G for efficient and selective C-to-T conversion at C-rich sequences. BMC Biol. 2021;19(1):34-34.
96	Z Liu, H Shan, S Chen, et al. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion. FASEB J. 2019;33(8):9210-9219.
97	W Jiang, S Feng, S Huang, et al. BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity. Cell Res. 2018;28(8):855. 1;-861;7.
98	AC Komor, KT Zhao, MS Packer, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:g-to-T:a base editors with higher efficiency and product purity. Sci Adv. 2017;3(8):eaao4774-eaao4774.
99	LW Koblan, JL Doman, C Wilson, et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol. 2018;36(9):843-848.
100	BW Thuronyi, LW Koblan, JM Levy, et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat Biotechnol. 2019;37(9):1070-1079.
101	C Zhou, Y Sun, R Yan, et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature (London). 2019;571(7764):275-278.
102	J Grünewald, R Zhou, S Iyer, et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol. 2019;37(9):1041-1048.
103	MF Richter, KT Zhao, E Eton, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020;38(7):883-891.
104	L Chen, S Zhang, N Xue, et al. Engineering a precise adenine base editor with minimal bystander editing. Nat Chem Biol. 2023;19(1):101-110.
105	Y-L Xiao, Y Wu, W Tang, An adenine base editor variant expands context compatibility. Nat Biotechnol. 2024.
106	L Chen, JE Park, P Paa, et al. Programmable C:g to G:c genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat Commun. 2021;12(1):1384-1384.
107	YK Jeong, S Lee, G-H Hwang, et al. Adenine base editor engineering reduces editing of bystander cytosines. Nat Biotechnol. 2021;39(11):1426-1433.
108	L Chen, B Zhu, G Ru, et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Nat Biotechnol. 2023;41(5):663-672.
109	N Sun, D Zhao, S Li, Z Zhang, C Bi, X Zhang. Reconstructed glycosylase base editors GBE2.0 with enhanced C-to-G base editing efficiency and purity. Mol Ther. 2022;30(7):2452-2463.
110	AV Anzalone, PB Randolph, JR Davis, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149-157.
111	PJ Chen, JA Hussmann, J Yan, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021;184(22):5635-5652.
112	JW Nelson, PB Randolph, SP Shen, et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. 2022;40(3):402-410.
113	G Zhang, Y Liu, S Huang, et al. Enhancement of prime editing via xrRNA motif-joined pegRNA. Nat Commun. 2022;13(1):1856-1856.
114	X Li, X Wang, W Sun, et al. Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes. J Mol Cell Biol. 2022;14(4).
115	Y Feng, S Liu, Q Mo, P Liu, X Xiao, H Ma. Enhancing prime editing efficiency and flexibility with tethered and split pegRNAs. Protein & cell. 2023;14(4):304-308.
116	Y Liu, G Yang, S Huang, et al. Enhancing prime editing by Csy4-mediated processing of pegRNA. Cell Res. 2021;31(10):1134-1136.
117	W Xu, Y Yang, B Yang, et al. A design optimized prime editor with expanded scope and capability in plants. Nat Plants. 2022;8(1):45-52.
118	X Li, L Zhou, BQ Gao, et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat Commun. 2022;13(1):1669-1669.
119	J Choi, W Chen, CC Suiter, et al. Precise genomic deletions using paired prime editing. Nat Biotechnol. 2022;40(2):218-226.
120	AV Anzalone, XD Gao, CJ Podracky, et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat Biotechnol. 2022;40(5):731-740.
121	T Jiang, X-O Zhang, Z Weng, W Xue. Deletion and replacement of long genomic sequences using prime editing. Nat Biotechnol. 2022;40(2):227-234.
122	J Kweon, H-Y Hwang, H Ryu, A-H Jang, D Kim, Y Kim. Targeted genomic translocations and inversions generated using a paired prime editing strategy. Mol Ther. 2023;31(1):249-259.
123	Y Jiao, M Li, X He, et al. Targeted, programmable, and precise tandem duplication in the mammalian genome. Genome Res. 2023;33(5):779-786.
124	MTN Yarnall, EI Ioannidi, C Schmitt-Ulms, et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat Biotechnol. 2023;41(4):500-512.
125	C Li, A Georgakopoulou, GA Newby, et al. In vivo HSC prime editing rescues sickle cell disease in a mouse model. Blood. 2023;141(17):2085-2099.
126	KA Everette, GA Newby, RM Levine, et al. Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice. Nature Biomedical Engineering. 2023;7(5):616-628.
127	KM Esvelt, JC Carlson, DR Liu. A system for the continuous directed evolution of biomolecules. Nature (London). 2011;472(7344):499-503.
128	TB Roth, BM Woolston, G Stephanopoulos, DR Liu, Harvard Univ CMA. Phage-assisted evolution of Bacillus methanolicus methanol dehydrogenase 2. ACS Synth Biol. 2019;8(4):796-806.
129	JL Doman, S Pandey, ME Neugebauer, et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell. 2023;186(18):3983-4002.
130	D B?ck, T Rothgangl, L Villiger, et al. In vivo prime editing of a metabolic liver disease in mice. Sci Transl Med. 2022;14(636):eabl9238-eabl9238.
131	R Liang, Z He, KT Zhao, et al. Prime editing using CRISPR-Cas12a and circular RNAs in human cells. Nat Biotechnol. 2024.
132	J Yan, P Oyler-Castrillo, P Ravisankar, et al. Improving prime editing with an endogenous small RNA-binding protein. Nature (London). 2024;628(8008):639-647.
133	HK Kim, G Yu, J Park, et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat Biotechnol. 2021;39(2):198-206.
134	Y Li, J Chen, SQ Tsai, Y Cheng. Easy-Prime: a machine learning–based prime editor design tool. Genome Biol. 2021;22(1):235-235.
135	F Liu, S Huang, J Hu, et al. Design of prime-editing guide RNAs with deep transfer learning. Nature machine intelligence. 2023;5(11):1261-1274.
136	M Song, JM Lim, S Min, et al. Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain. Nat Commun. 2021;12(1):5617-5617.
137	Q Lin, S Jin, Y Zong, et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat Biotechnol. 2021;39(8):923-927.
138	Y Zhuang, J Liu, H Wu, et al. Increasing the efficiency and precision of prime editing with guide RNA pairs. Nat Chem Biol. 2022;18(1):29-37.
139	B Zetsche, S Gootenberg Jonathan, O Abudayyeh Omar, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759-771.
140	JS Chen, E Ma, LB Harrington, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360(6387):436-439.
141	SR Rananaware, EK Vesco, GM Shoemaker, et al. Programmable RNA detection with CRISPR-Cas12a. Nat Commun. 2023;14(1):5409-5409.
142	G Mao, X Luo, S Ye, et al. Fluorescence and colorimetric analysis of African Swine fever virus based on the RPA-assisted CRISPR/Cas12a strategy. Analytical chemistry (Washington). 2023;95(20):8063-8069.
143	X Ling, L Chang, H Chen, et al. Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates. Mol Cell. 2021;81(22):4747-4756.
144	LB Harrington, D Burstein, JS Chen, et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science. 2018;362(6416):839-842.
145	T Karvelis, G Bigelyte, JK Young, et al. PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Res. 2020;48(9):5016-5023.
146	Z Wu, Y Zhang, H Yu, et al. Programmed genome editing by a miniature CRISPR-Cas12f nuclease. Nat Chem Biol. 2021;17(11):1132-1138.
147	X Kong, H Zhang, G Li, et al. Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing. Nat Commun. 2023;14(1):2046-2046.
148	L Gao, DBT Cox, WX Yan, et al. Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol. 2017;35(8):789-792.
149	M Tu, L Lin, Y Cheng, et al. A new lease of life': fnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res. 2017;45(19):11295-11304.
150	BP Kleinstiver, AA Sousa, RT Walton, et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene. Nat Biotechnol. 2019;37(3):276-282.
151	Y Wu, Q Yuan, Y Zhu, X Gao, J Song, Z Yin. Improving FnCas12a genome editing by exonuclease fusion. CRISPR J. 2020;3(6):503-511.
152	L Zhang, JA Zuris, R Viswanathan, et al. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat Commun. 2021;12(1):3908-3908.
153	X Liu, X Liu, C Zhou, et al. Engineered FnCas12a with enhanced activity through directional evolution in human cells. J Biol Chem. 2021;296:100394.
154	X Xu, A Chemparathy, L Zeng, et al. Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol Cell. 2021;81(20):4333-4345.
155	DY Kim, JM Lee, SB Moon, et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat Biotechnol. 2022;40(1):94-102.
156	Y Wang, Y Wang, D Pan, et al. Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease. Cell reports (Cambridge). 2022;40(13):111418-111418.
157	T Wu, C Liu, S Zou, et al. An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity. Nat Chem Biol. 2023;19(11):1384-1393.
158	Z Wu, D Liu, D Pan, et al. Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1. Nature catalysis. 2023;6(8):695-709.
159	M Su, F Li, Y Wang, et al. Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity. Nat Chem Biol. 2023.
160	T Hino, SN Omura, R Nakagawa, et al. An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis. Cell. 2023;186(22):4920-4935.
161	P Siguier, E Gourbeyre, M Chandler. Bacterial insertion sequences: their genomic impact and diversity. FEMS Microbiol Rev. 2014;38(5):865-891.
162	VV Kapitonov, KS Makarova, EV Koonin. ISC, a novel group of bacterial and archaeal DNA transposons that encode Cas9 homologs. J Bacteriol. 2016;198(5):797-807.
163	H Altae-Tran, S Kannan, FE Demircioglu, et al. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science. 2021;374(6563):57-65.
164	T Karvelis, G Druteika, G Bigelyte, et al. Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature (London). 2021;599(7886):692-696.
165	G Xiang, Y Li, J Sun, et al. Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors. Nat Biotechnol. 2023;
166	D Han, Q Xiao, Y Wang, et al. Development of miniature base editors using engineered IscB nickase. Nat Methods. 2023;20(7):1029-1036.
167	S Shmakov, O Abudayyeh Omar, S Makarova Kira, et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol Cell. 2015;60(3):385-397.
168	OO Abudayyeh, JS Gootenberg, S Konermann, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016;353(6299):557-557.
169	A East-Seletsky, MR O'Connell, SC Knight, et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature. 2016;538(7624):270-273.
170	AA Smargon, DBT Cox, NK Pyzocha, et al. Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Mol Cell. 2017;65(4):618-630.
171	S Konermann, P Lotfy, NJ Brideau, J Oki, MN Shokhirev, PD Hsu. Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors. Cell. 2018;173(3):665-676.
172	JS Gootenberg, OO Abudayyeh, JW Lee, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438-442.
173	CA Freije, C Myhrvold, CK Boehm, et al. Programmable inhibition and detection of RNA viruses using Cas13. Mol Cell. 2019;76(5):826-837.
174	L-Z Yang, Y Wang, S-Q Li, et al. Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems. Mol Cell. 2019;76(6):981-997.
175	B He, W Peng, J Huang, et al. Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. Protein & cell. 2020;11(7):518-524.
176	C Zhou, X Hu, C Tang, et al. CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration. Natl Sci Rev. 2020;7(5):835-837.
177	BY Mok, MH de Moraes, J Zeng, et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature (London). 2020;583(7817):631-637.
178	S-I Cho, S Lee, YG Mok, et al. Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell. 2022;185(10):1764-1776.
179	SR Bacman, SL Williams, M Pinto, S Peralta, CT Moraes. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nat Med. 2013;19(9):1111-1113.
180	PA Gammage, J Rorbach, AI Vincent, EJ Rebar, M Minczuk. Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations. EMBO Mol Med. 2014;6(4):458-466.
181	Z Yi, X Zhang, W Tang, et al. Strand-selective base editing of human mitochondrial DNA using mitoBEs. Nat Biotechnol. 2023;
182	M Saito, P Xu, G Faure, et al. Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature. 2023.
183	H Altae-Tran, S Kannan, AJ Suberski, et al. Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering. Science. 2023;382(6673):eadi1910-eadi1910.
184	ZX Liu, S Zhang, HZ Zhu, et al. Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage. Science. 2024;383(6682):383-495.
185	CR Conti. Re-thinking angina. Clinical cardiol (Mahwah, NJ). 2007;30(1). I-1-I-3.
186	C Müller. Xanthomata, hypercholesterolemia, pectoris angina. Acta Med Scand. 1938;95(S89):75-84.
187	LJ Goldstein, SM Brown. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46(1):897-930.
188	HB Hubert, M Feinleib, PM McNamara, WP Castelli. Obesity as an independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the Framingham Heart Study. Circulation (New York, NY). 1983;67(5):968-977.
189	AP Monaco, RL Neve, C Colletti-Feener, CJ Bertelson, DM Kurnit, LM Kunkel. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature (London). 1986;323(6089):646-650.
190	P Moulin, L Wickham, E Bruckert, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet. 2003;34(2):154-156.
191	I Postmus, S Trompet, HA Deshmukh, et al. Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins. Nat Commun. 2014;5(1):5068.
192	GF Watts, C Schwabe, R Scott, et al. RNA interference targeting ANGPTL3 for triglyceride and cholesterol lowering: phase 1 basket trial cohorts. Nat Med. 2023;29(9):2216-2223.
193	AW Turner, D Wong, CN Dreisbach, CL Miller. GWAS reveal targets in vessel wall pathways to treat coronary artery disease. Front Cardiovasc Med. 2018;5:72.
194	TS Roman, KL Mohlke. Functional genomics and assays of regulatory activity detect mechanisms at loci for lipid traits and coronary artery disease. Curr Opin Genet Dev. 2018;50:52-59.
195	AJ Smith, SE Humphries, PJ Talmud. Identifying functional noncoding variants from genome-wide association studies for cardiovascular disease and related traits. Curr Opin Lipidol. 2015;26(2):120-126.
196	G Runmin, J Jiamei, J Zhiliang, et al. Genetic variation of CXCR4 and risk of coronary artery disease: epidemiological study and functional validation of CRISPR/Cas9 system. Oncotarget. 2018;9(18):14077-14083.
197	MM Nagiec, AP Skepner, J Negri, et al. Modulators of hepatic lipoprotein metabolism identified in a search for small-molecule inducers of tribbles pseudokinase 1 expression. PLoS One. 2015;10(3):e0120295.
198	S Lalonde, VA Codina-Fauteux, SM de Bellefon, et al. Integrative analysis of vascular endothelial cell genomic features identifies AIDA as a coronary artery disease candidate gene. Genome Biol. 2019;20(1):133.
199	MD Krause, RT Huang, D Wu, et al. Genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics. Proc Natl Acad Sci USA. 2018;115(48):E11349-E11358.
200	H Yu, A Rimbert, AE Palmer, et al. GPR146 deficiency protects against hypercholesterolemia and atherosclerosis. Cell. 2019;179(6):1276-1288.
201	Y Wang, L Chen, Z Tian, et al. CRISPR-Cas9 mediated gene knockout in human coronary artery endothelial cells reveals a pro-inflammatory role of TLR2. Cell Biol Int. 2018;42(2):187-193.
202	Q Hai, B Ritchey, P Robinet, et al. Quantitative trait locus mapping of macrophage cholesterol metabolism and CRISPR/Cas9 editing implicate an ACAT1 truncation as a causal modifier variant. Arterioscler Thromb Vasc Biol. 2017;38(1):83-91.
203	CA Castellani, RJ Longchamps, JA Sumpter, et al. Mitochondrial DNA copy number can influence mortality and cardiovascular disease via methylation of nuclear DNA CpGs. Genome Med. 2020;12(1):84.
204	BL Clifford, KE Jarrett, J Cheng, et al. RNF130 regulates LDLR availability and plasma LDL cholesterol levels. Circ Res. 2023;132(7):849-863.
205	A Maillet, K Tan, X Chai, et al. Modeling doxorubicin-induced cardiotoxicity in human pluripotent stem cell derived-cardiomyocytes. Sci Rep. 2016;6(1):25333.
206	S Chatterjee, T Hofer, A Costa, et al. Telomerase therapy attenuates cardiotoxic effects of doxorubicin. Mol Ther. 2021;29(4):1395-1410.
207	M Vrablik, D Dlouha, V Todorovova, D Stefler, JA Hubacek. Genetics of cardiovascular disease: how far are we from personalized cvd risk prediction and management? Int J Mol Sci. 2021;22(8):4182.
208	R Liu, L Liang, EF Freed, RT Gill. Directed evolution of CRISPR/Cas systems for precise gene editing. Trends Biotechnol. 2021;39(3):262-273.
209	JPK Bravo, M-S Liu, GN Hibshman, et al. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature (London). 2022;603(7900):343-347.
210	J Huang, Q Lin, H Fei, et al. Discovery of deaminase functions by structure-based protein clustering. Cell. 2023;
211	Y-r Cui, S-j Wang, T Ma, et al. KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export. Commun Biol. 2022;5(1):237-237.
212	Z Liu, S Chen, L Lai, Z Li. Inhibition of base editors with anti-deaminases derived from viruses. Nat Commun. 2022;13(1):597-597.
213	L Chen, S Zhang, N Xue, et al. Engineering a precise adenine base editor with minimal bystander editing. Nat Chem Biol. 2023;19(1):101.
214	C Wang, Y Qu, JKW Cheng, et al. dCas9-based gene editing for cleavage-free genomic knock-in of long sequences. Nat Cell Biol. 2022.
215	M Kawamata, HI Suzuki, R Kimura, A Suzuki. Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs. Nat Biomed Eng. 2023;7(5):672-691.
216	S Zhang, J Shen, D Li, Y Cheng. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing. Theranostics. 2021;11(2):614-648.
217	L Li, S Hu, X Chen. Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities. Biomaterials. 2018;171:207-218.
218	D Wang, F Zhang, G Gao. CRISPR-based therapeutic genome editing: strategies and in vivo delivery by AAV vectors. Cell. 2020;181(1):136-150.
219	V Madigan, Y Zhang, R Raghavan, et al. Human paraneoplastic antigen Ma2 (PNMA2) forms icosahedral capsids that can be engineered for mRNA delivery. Proc Natl Acad Sci U S A. 2024;121(11):e2307812120.
220	S Dubey, Z Chen, YJ Jiang, et al. Small extracellular vesicles (sEVs)-based gene delivery platform for cell-specific CRISPR/Cas9 genome editing. Theranostics. 2024;14(7):2777-2793.
221	W Tang, T Tong, H Wang, et al. A DNA origami-based gene editing system for efficient gene therapy in vivo. Angewandte Chemie (International ed). 2023;62(51):e202315093.
222	JL Katzmann, AJ Cupido, U Laufs. Gene therapy targeting PCSK9. Metabolites. 2022;12(1).
223	<A CRISPR edit for heart disease.pdf>.
224	Y Wu, L Zhou, X Wang, et al. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A. Cell Discov. 2016;2(1):16014-16014.
225	F-C Zhu, X-H Guan, Y-H Li, et al. Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet. 2020;396(10249):479-488.
226	Q Ding, A Strong, KM Patel, et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res. 2014;115(5):488-492.
227	X Wang, A Raghavan, T Chen, et al. CRISPR-Cas9 targeting of PCSK9 in human hepatocytes in vivo—brief report. Arterioscler Thromb Vasc Biol. 2016;36(5):783-786.
228	L Xu, KH Park, L Zhao, et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol Ther. 2016;24(3):564-569.
229	AC Chadwick, NH Evitt, W Lv, K Musunuru. Reduced blood lipid levels with in vivo CRISPR-Cas9 base editing of ANGPTL3. Circulation. 2018;137(9):975-977.
230	X Gu, R Chai, L Guo, et al. Transduction of adeno-associated virus vectors targeting hair cells and supporting cells in the neonatal mouse cochlea. Front cell neurosci. 2019;13:8.
231	J Ai, J Li, Q Su, et al. rAAV-delivered PTEN therapeutics for prostate cancer. Molecular therapy Nucleic acids. 2022;27:122-132.
232	FA Ran, L Cong, WX Yan, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature (London). 2015;520(7546):186-191.
233	C Long, L Amoasii, AA Mireault, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351(6271):400-403.
234	CE Nelson, CH Hakim, DG Ousterout, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403-407.
235	Y Zhang, H Li, Y-L Min, et al. Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system. Sci Adv. 2020;6(8):eaay6812-eaay6812.
236	F Chemello, AC Chai, H Li, et al. Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing. Sci Adv. 2021;7(18).
237	X Pan, L Philippen, SK Lahiri, et al. In vivo Ryr2 editing corrects catecholaminergic polymorphic ventricular tachycardia. Circ Res. 2018;123(8):953-963.
238	H Zhao, Y Li, L He, et al. In vivo AAV-CRISPR/Cas9-mediated gene editing ameliorates atherosclerosis in familial hypercholesterolemia. Circulation. 2020;141(1):67-79.
239	Z Liu, S Chen, W Xie, et al. Versatile and efficient in vivo genome editing with compact Streptococcus pasteurianus Cas9. Mol Ther. 2022;30(1):256-267.
240	J Dave, N Raad, N Mittal, et al. Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact. Cardiovasc Res. 2022;118(15):3140-3150.
241	T Nishiyama, Y Zhang, M Cui, et al. Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy. Sci Transl Med. 2022;14(672):eade1633-eade1633.
242	H Zhang. A bright future for lipid nanoparticles in gene therapy. Cell and Gene Therapy Insights. 2021;7(6):755-758.
243	JD Finn, AR Smith, MC Patel, et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell reports (Cambridge). 2018;22(9):2227-2235.
244	L Zhang, L Wang, Y Xie, et al. Triple-targeting delivery of CRISPR/Cas9 to reduce the risk of cardiovascular diseases. Angew Chem Int Ed Engl. 2019;58(36):12404-12408.
245	J Gong, HX Wang, YH Lao, et al. A versatile nonviral delivery system for multiplex gene-editing in the liver. Adv Mater. 2020;32(46).
246	M Qiu, Z Glass, J Chen, et al. Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3. Proc Natl Acad Sci USA. 2021;118(10).
247	E Kenjo, H Hozumi, Y Makita, et al. Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice. Nat Commun. 2021;12(1):7101-7113.
248	S Banskota, A Raguram, S Suh, et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell. 2022;185(2):250-265.
249	K Musunuru, AC Chadwick, T Mizoguchi, et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature. 2021;593(7859):429-434.
250	T Rothgangl, MK Dennis, PJC Lin, et al. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat Biotechnol. 2021;39(8):949-957.
251	LN Kasiewicz, S Biswas, A Beach, et al. GalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy. Nat Commun. 2023;14(1):2776.
252	C Arnold, P Webster. 11 clinical trials that will shape medicine in 2024. Nat Med. 2023;29(12):2964-2968.
253	JD Gillmore, ML Maitland, D Lebwohl. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N Engl J Med. 2021;385(18):1722-1723. Reply..
254	A Lek, B Wong, A Keeler, et al. Death after high-dose rAAV9 gene therapy in a patient with Duchenne's muscular dystrophy. N Engl J Med. 2023;389(13):1203-1210.
255	CT Charlesworth, PS Deshpande, DP Dever, et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 2019;25(2):249-254.
256	A Kapelanski-Lamoureux, Z Chen, Z-H Gao, et al. Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma. Mol Ther. 2022;30(12):3542.
257	Z Aherrahrou, S Schlossarek, S Stoelting, et al. Knock-out of nexilin in mice leads to dilated cardiomyopathy and endomyocardial fibroelastosis. Basic research in cardiology. 2016;111(1):1-10.