Programmable Multiplex Genome Editing: Innovations in CRISPR Effectors, crRNA Engineering, and Delivery Strategies

Abstract

The discovery of CRISPR based technologies has transformed genome engineering and synthetic biology. With advancements in the ability to do multiplex genome editing, it is now emerging as an ideal approach for trait stacking to improve crops, functional genomics, and complex metabolic engineering in various biological systems. This review discusses engineering and optimization of the latest CRISPR effectors for scalable and precise multiplex editing, ranging from well-known systems like Cas9 and Cas12 variants, to newer, smaller variants such as CasMINI, Cas12j2, and Cas12k. We highlight how the emergence of base editors and prime editors enabled efficient editing across multiple loci without double strand breaks. We also elaborate on the expression and processing strategies of crRNA arrays, which are central to any multiplexing approach. These include tRNA-based and ribozyme-mediated methods, synthetic modular designs, and AI-optimized guide RNAs tailored to diverse systems. Additionally, we assess next-generation delivery platforms such as lipid nanoparticles, virus-like particles, and metal-organic frameworks that overcome conventional barriers in in vivo applications. This review provides a critical take on technological advances enabling precise, high-throughput, and programmable multiplex genome editing across biological systems, setting the foundation for future innovations in synthetic biology, crop improvement, and therapeutic intervention in multigene diseases.

Keywords

CRISPR / Cas / Multiplex genome editing / crRNA array / Cas12 variants / Delivery platforms / Modular CRISPR constructs / Crop improvement

Acknowledgments

We thank Smruti Ranjan Panda for assistance in the preparation of the manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions

Conceptualization, D.R.; Writing—Original Draft Preparation, D.R. and G.B.; Writing—Review & Editing, D.R. and G.B.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No data was used for the research described in the article.

Funding

This research received no external funding.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

[1] Pickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 2019, 20, 490-507. doi:10.1038/s41580-019-0131-5.

[2] Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science 2014, 346, 1258096. doi:10.1126/science.1258096.

[3] Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013, 8, 2281-2308. doi:10.1038/nprot.2013.143.

[4] Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends Genet. 2021, 37, 639-656. doi:10.1016/j.tig.2021.02.008.

[5] Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas 9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 2015, 33, 538-542. doi:10.1038/nbt.3190.

[6] Reuven N, Adler J, Broennimann K, Myers N, Shaul Y. Recruitment of DNA repair MRN complex by intrinsically disordered protein domain fused to Cas 9 improves efficiency of CRISPR-mediated genome editing. Biomolecules 2019, 9, 584. doi:10.3390/biom9100584.

[7] Li G, Zhang X, Wang H, Liu D, Li Z, Wu Z, et al. Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors. Int. J. Biochem. Cell Biol. 2020, 125, 105790. doi:10.1016/j.biocel.2020.105790.

[8] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013, 339, 819-823. doi:10.1126/science.1231143.

[9] Zhang Y, Buchholz F, Muyrers JP, Stewart AF. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 1998, 20, 123-128. doi:10.1038/2417.

[10] Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 2009, 460, 894-898. doi:10.1038/nature08187.

[11] Bao Z, Cobb RE, Zhao H.Accelerated genome engineering through multiplexing. Wiley Interdiscip Rev. Syst. Biol. Med. 2016, 8, 5-21. doi:10.1002/wsbm.1319.

[12] Barbieri EM, Muir P, Akhuetie-Oni BO, Yellman CM, Isaacs FJ. Precise editing at DNA replication forks enables multiplex genome engineering in eukaryotes. Cell 2017, 171, 1453-1467.e13. doi:10.1016/j.cell.2017.10.034.

[13] Ronda C, Pedersen LE, Sommer MO, Nielsen AT.CRMAGE: CRISPR optimized mage recombineering. Sci. Rep. 2016, 6, 19452. doi:10.1038/srep19452.

[14] Nyerges Á, Csörgő B, Nagy I, Bálint B, Bihari P, Lázár V, et al. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proc. Nat. Acad. Sci. USA 2016, 113, 2502-2507, Mar. doi:10.1073/pnas.1520040113.

[15] García-Otín AL, Guillou F. Mammalian genome targeting using site-specific recombinases. Front. Biosci. 2006, 11, 1108-1136.

[16] Tian X, Zhou B. Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution. J. Biol. Chem. 2021, 296, doi:10.1016/j.jbc.2021.100509.

[17] Durrant MG, Perry NT, Pai JJ, Jangid AR, Athukoralage JS, Hiraizumi M, et al. Bridge RNAs direct programmable recombination of target and donor DNA. Nature 2024, 630, 984-993. doi:10.1038/s41586-024-07552-4.

[18] Hiraizumi M, Perry NT, Durrant MG, Soma T, Nagahata N, Okazaki S, et al. Structural mechanism of bridge RNA-guided recombination. Nature 2024, 630, 994-1002. doi:10.1038/s41586-024-07570-2.

[19] Partridge SR, Hall RM. The IS 1111 family members IS 4321 and IS 5075 have subterminal inverted repeats and target the terminal inverted repeats of Tn 21 family transposons. J. Bacteriol. 2003, 185, 6371-6384. doi:10.1128/JB.185.21.6371-6384.2003.

[20] Choi S, Ohta S, Ohtsubo E. A novel IS element, IS 621, of the IS 110/IS 492 family transposes to a specific site in repetitive extragenic palindromic sequences in Escherichia coli. J. Bacteriol. 2003, 185, 4891-4900. doi:10.1128/JB.185.16.4891-4900.2003.

[21] Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013, 31, 397-405. doi:10.1016/j.tibtech.2013.04.004.

[22] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816-821. doi:10.1126/science.1225829.

[23] Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies. Cell 2024, 187, 1076-1100. doi:10.1016/j.cell.2024.01.042.

[24] Yin L, Xi D, Shen Y, Ding N, Shao Q, Qian Y, et al. Rewiring metabolic flux in Corynebacterium glutamicum using a CRISPR/dCpf1-based bifunctional regulation system. J. Agric. Food Chem. 2024, 72, 3077-3087. doi:10.1021/acs.jafc.3c08529.

[25] Lim SR, Lee HJ, Kim HJ, Lee SJ. Multiplex single-nucleotide microbial genome editing achieved by CRISPR-Cas9 using 5′-end-truncated sgRNAs. ACS Synth. Biol. 2023, 12, 2203-2207. doi:10.1021/acssynbio.3c00323.

[26] Utomo JC, Hodgins CL, Ro DK. Multiplex genome editing in yeast by CRISPR/Cas9-a potent and agile tool to reconstruct complex metabolic pathways. Front. Plant Sci. 2021, 12, 719148. doi:10.3389/fpls.2021.719148.

[27] Ronda C, Maury J, Jakočiu̅nas T, Baallal Jacobsen SA, Germann SM, Harrison SJ, et al. CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae. Microb. Cell Factor. 2015, 14, 97. doi:10.1186/s12934-015-0288-3.

[28] Lorenzo CD, Debray K, Herwegh D, Develtere W, Impens L, Schaumont D, et al. BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize. Plant Cell 2023, 35, 218-238. doi:10.1093/plcell/koac243.

[29] Ni P, Zhao Y, Zhou X, Liu Z, Huang Z, Ni Z, et al. Efficient and versatile multiplex prime editing in hexaploid wheat. Genome Biol. 2023, 24, 156. doi:10.1186/s13059-023-02990-1.

[30] Bai M, Yuan J, Kuang H, Gong P, Li S, Zhang Z, et al. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas 9 in soya bean. Plant Biotechnol. J. 2020, 18, 721-731. doi:10.1111/pbi.13239.

[31] Lu Y, Ye X, Guo R, Huang J, Wang W, Tang J, et al. Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system. Mol. Plant 2017, 10, 1242-1245. doi:10.1016/j.molp.2017.06.007.

[32] Jacobs TB, Zhang N, Patel D, Martin GB. Generation of a collection of mutant tomato lines using pooled CRISPR libraries. Plant Physiol. 2017, 174, 2023-2037. doi:10.1104/pp.17.00489.

[33] Han K, Jeng EE, Hess GT, Morgens DW, Li A, Bassik MC. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat. Biotechnol. 2017, 35, 463-474. doi:10.1038/nbt.3834.

[34] Gao Z, Fan M, Das AT, Herrera-Carrillo E, Berkhout B. Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA. Nucleic Acids Res. 2020, 48, 5527-5539. doi:10.1093/nar/gkaa226.

[35] Thompson DB, Aboulhouda S, Hysolli E, Smith CJ, Wang S, Castanon O, et al. The Future of Multiplexed Eukaryotic Genome Engineering. ACS Chem. Biol. 2018, 13, 313-325. doi:10.1021/acschembio.7b00842.

[36] Yates KB, Tonnerre P, Martin GE, Gerdemann U, Al Abosy R, Comstock DE, et al. Epigenetic scars of CD8+ T cell exhaustion persist after cure of chronic infection in humans. Nat. Immunol. 2021, 22, 1020-1029. doi:10.1038/s41590-021-00979-1.

[37] Tang K, Zhou L, Tian X, Fang SY, Vandenbulcke E, Du A, et al. Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering. Nat. Biomed. Eng. 2025. doi:10.1038/s41551-025-01371-2.

[38] Guo S, Gao G, Zhang C, Peng G. Multiplexed genome editing for efficient phenotypic screening in Zebrafish. Vet. Sci. 2022, 9, 92. doi:10.3390/vetsci9020092.

[39] Singh J, Sharma D, Brar GS, Sandhu KS, Wani SH, Kashyap R, et al. CRISPR/Cas tool designs for multiplex genome editing and its applications in developing biotic and abiotic stress-resistant crop plants. Mol. Biol. Rep. 2022, 49, 11443-11467. doi:10.1007/s11033-022-07741-2.

[40] Abdelrahman M, Wei Z, Rohila JS, Zhao K. Multiplex genome-editing technologies for revolutionizing plant biology and crop improvement. Front. Plant Sci. 2021, 12, 721203. doi:10.3389/fpls.2021.721203.

[41] Hu Y, Patra P, Pisanty O, Shafir A, Belew ZM, Binenbaum J, et al. Multi-Knock—a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants. Nat. Plants 2023, 9, 572-587. doi:10.1038/s41477-023-01374-4.

[42] Adiego-Perez B, Randazzo P, Daran JM, Verwaal R, Roubos JA, Daran-Lapujade P, et al. Multiplex genome editing of microorganisms using CRISPR-Cas. FEMS Microbiol. Lett. 2019, 366, fnz086. doi:10.1093/femsle/fnz086.

[43] Lim SR, Lee SJ. Multiplex CRISPR-Cas genome editing: next-generation microbial strain engineering. J. Agr. Food Chem. 2024, 72, 11871-11884. doi:10.1021/acs.jafc.4c01650.

[44] Alok A, Sandhya D, Jogam P, Rodrigues V, Bhati KK, Sharma H, et al. The rise of the CRISPR/Cpf1 system for efficient genome editing in plants. Front. Plant Sci. 2020, 11, 264. doi:10.3389/fpls.2020.00264.

[45] Paul B, Montoya G. CRISPR-Cas12a: Functional overview and applications. Biomed. J. 2020, 43, 8-17. doi:10.1016/j.bj.2019.10.005.

[46] Walton RT, Christie KA, Whittaker MN, Kleinstiver BP. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 2020, 368, 290-296. doi:10.1126/science.aba8853.

[47] Nishimasu H, Cong L, Yan WX, Ran FA, Zetsche B, Li Y, et al. Crystal structure of Staphylococcus aureus Cas9. Cell 2015, 162, 1113-1126. doi:10.1016/j.cell.2015.08.007.

[48] Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 2015, 520, 186-191. doi:10.1038/nature14299.

[49] Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 2014, 343, 1247997. doi:10.1126/science.1247997.

[50] Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med. 2018, 24, 1216-1224. doi:10.1038/s41591-018-0137-0.

[51] Agudelo D, Carter S, Velimirovic M, Duringer A, Rivest JF, Levesque S, et al. Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9. Genome Res. 2020, 30, 107-117. doi:10.1101/gr.255414.119.

[52] Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015, 163, 759-771. doi:10.1016/j.cell.2015.09.038.

[53] Hino T, Omura SN, Nakagawa R, Togashi T, Takeda SN, Hiramoto T, et al. An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis. Cell 2023, 186, 4920-4935. doi:https://doi.org/10.1016/j.cell.2023.08.031.

[54] Zhang L, Zuris JA, Viswanathan R, Edelstein JN, Turk R, Thommandru B, et al. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat. Commun. 2021, 12, 3908. doi:10.1038/s41467-021-24017-8.

[55] Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 2019, 37, 276-282. doi:10.1038/s41587-018-0011-0.

[56] Hu Y, Han L, Mo Q, Du Z, Jiang W, Wu X, et al. Engineering miniature CRISPR-Cas Un1Cas12f1 for efficient base editing. Mol. Ther. Nucleic Acids 2024, 35, 102201. doi:10.1016/j.omtn.2024.102201.

[57] Chen L, Zhou X, Huang C, Zhang Y, Xin C, Hong J, et al. Engineered Un1Cas12f1 with boosted gene-editing activity and expanded genomic coverage. Proc. Nat. Acad. Sci. USA 2025, 122, e2501292122. doi:10.1073/pnas.2501292122.

[58] Xu X, Chemparathy A, Zeng L, Kempton HR, Shang S, Nakamura M, et al. Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol. Cell 2021, 81, 4333-4345. doi:https://doi.org/10.1016/j.molcel.2021.08.008.

[59] Pausch P, Soczek KM, Herbst DA, Tsuchida CA, Al-Shayeb B, Banfield JF, et al. DNA interference states of the hypercompact CRISPR-CasΦ effector. Nat. Struct. Mol. Biol. 2021, 28, 652-661. doi:10.1038/s41594-021-00632-3.

[60] Pausch P, Al-Shayeb B, Bisom-Rapp E, Tsuchida CA, Li Z, Cress BF, et al. CRISPR-CasΦ from huge phages is a hypercompact genome editor. Science 2020, 369, 333-337. doi:10.1126/science.abb1400.

[61] Al-Shayeb B, Skopintsev P, Soczek KM, Stahl EC, Li Z, Groover E, et al. Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors. Cell 2022, 185, 4574-486. doi:10.1016/j.cell.2022.10.020.

[62] Gaudelli NM, Lam DK, Rees HA, Solá-Esteves NM, Barrera LA, Born DA, et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 2020, 38, 892-900. doi:10.1038/s41587-020-0491-6.

[63] Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016, 533, 420-424. doi:10.1038/nature17946.

[64] Neugebauer ME, Hsu A, Arbab M, Krasnow NA, McElroy AN, Pandey S, et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat. Biotechnol. 2023, 41, 673-685. doi:10.1038/s41587-022-01533-6.

[65] Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 2016, 353, aaf8729. doi:10.1126/science.aaf8729.

[66] Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 2020, 38, 883-891. doi:10.1038/s41587-020-0453-z.

[67] Anzalone AV, Gao XD, Podracky CJ, Nelson AT, Koblan LW, Raguram A, et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 2022, 40, 731-740. doi:10.1038/s41587-021-01133-w.

[68] Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019, 576, 149-157. doi:10.1038/s41586-019-1711-4.

[69] Yan J, Oyler-Castrillo P, Ravisankar P, Ward CC, Levesque S, Jing Y, et al. Improving prime editing with an endogenous small RNA-binding protein. Nature 2024, 628, 639-647. doi:10.1038/s41586-024-07259-6.

[70] Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 2021, 184, 5635-5652. doi:10.1016/j.cell.2021.09.018.

[71] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013, 339, 823-826. doi:10.1126/science.1232033.

[72] Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 2013, 31, 233-239. doi:10.1038/nbt.2508.

[73] Otoupal PB, Ito M, Arkin AP, Magnuson JK, Gladden JM, Skerker JM. Multiplexed CRISPR-Cas9-based genome editing of Rhodosporidium toruloides. Msphere 2019, 4, 10-128. doi:10.1128/msphere.00099-19.

[74] Sánchez-León S, Marín-Sanz M, Guzmán-López MH, Gavilán-Camacho M, Simón E, Barro F. CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins: paving the way for gliadin-free wheat. J. Exp. Botany 2024, 75, 7079-7095. doi:10.1093/jxb/erae376.

[75] Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 2017, 357, 1303-1307. doi:10.1126/science.aan4187.

[76] Ratan ZA, Son YJ, Haidere MF, Uddin BM, Yusuf MA, Zaman SB, et al. CRISPR-Cas9: a promising genetic engineering approach in cancer research. Ther. Adv. Med. Oncol. 2018, 10, 1758834018755089. doi:10.1177/1758834018755089.

[77] Rabaan AA, AlSaihati H, Bukhamsin R, Bakhrebah MA, Nassar MS, Alsaleh AA, et al. Application of CRISPR/Cas9 technology in cancer treatment: a future direction. Curr. Oncol. 2023, 30, 1954-1976. doi:10.3390/curroncol30020152.

[78] Chehelgerdi M, Chehelgerdi M, Khorramian-Ghahfarokhi M, Shafieizadeh M, Mahmoudi E, Eskandari F, et al. Comprehensive review of CRISPR-based gene editing: mechanisms, challenges, and applications in cancer therapy. Mol. Cancer 2024, 23, 9. doi:10.1186/s12943-023-01925-5.

[79] Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 2017, 8, 14500. doi:10.1038/ncomms14500.

[80] Schmidt MJ, Gupta A, Bednarski C, Gehrig-Giannini S, Richter F, Pitzler C, et al. Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases. Nat. Commun. 2021, 12, 4219. doi:10.1038/s41467-021-24454-5.

[81] Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas 9 nucleases with improved specificity. Science 2016, 351, 84-88. doi:10.1126/science.aad5227.

[82] Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 2016, 529, 490-495. 2016, doi:10.1038/nature16526.

[83] Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 2017, 550, 407-410. doi:10.1038/nature24268.

[84] Huang X, Yang D, Zhang J, Xu J, Chen YE. Recent advances in improving gene-editing specificity through CRISPR-Cas9 nuclease engineering. Cells 2022, 11, 2186. doi:10.3390/cells11142186.

[85] Wang JY, Doudna JA. CRISPR technology: A decade of genome editing is only the beginning. Science 2023, 379, eadd8643. doi:10.1126/science.add8643.

[86] Bayat H, Modarressi MH, Rahimpour A. The conspicuity of CRISPR-Cpf1 system as a significant breakthrough in genome editing. Curr. Microbiol. 2018, 75, 107-115.

[87] Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 2017, 35, 31-34. doi:10.1038/nbt.3737.

[88] Li H, Zhu Z, Li S, Li J, Yan L, Zhang C, et al. Multiplex precision gene editing by a surrogate prime editor in rice. Mol. Plant 2022, 15, 1077-1080. doi:https://doi.org/10.1016/j.molp.2022.05.009.

[89] Zhang Y, Ren Q, Tang X, Liu S, Malzahn AA, Zhou J, et al. Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems. Nat. Commun. 2021, 12, 1944. doi:10.1038/s41467-021-22330-w.

[90] Li T, Zhu L, Xiao B, Gong Z, Liao Q, Guo J. CRISPR-Cpf1-mediated genome editing and gene regulation in human cells. Biotechnol. Adv. 2019, 37, 21-27. doi:https://doi.org/10.1016/j.biotechadv.2018.10.013.

[91] Pardue MT, Allen RS.Neuroprotective strategies for retinal disease. Progr. Retinal Eye Res. 2018, 65, 50-76. doi:https://doi.org/10.1016/j.preteyeres.2018.02.002.

[92] Sepah YJ, Velez G, Tang PH, Yang J, Chemudupati T, Li AS, et al. Proteomic analysis of intermediate uveitis suggests myeloid cell recruitment and implicates IL-23 as a therapeutic target. Am. J. Ophthalmol. Case Rep. 2020, 18, 100646. doi:https://doi.org/10.1016/j.ajoc.2020.100646.

[93] Bigini F, Lee SH, Sun YJ, Sun Y, Mahajan VB. Unleashing the potential of CRISPR multiplexing: Harnessing Cas12 and Cas 13 for precise gene modulation in eye diseases. Vision Res. 2023, 213, 108317. doi:https://doi.org/10.1016/j.visres.2023.108317.

[94] Xuan Q, Wang J, Nie Y, Fang C, Liang W. Research progress and application of miniature CRISPR-Cas 12 system in gene editing. Int. J. Mol. Sci. 2024, 25, 12686. doi:10.3390/ijms252312686.

[95] Wu H, Sun Y, Wang Y, Luo L, Song Y. Advances in miniature CRISPR-Cas proteins and their applications in gene editing. Arch. Microbiol. 2024, 206, 231. doi:10.1007/s00203-024-03962-0.

[96] Chen P, Zhou J, Wan Y, Liu H, Li Y, Liu Z, et al. A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing. Genome Biol. 2020, 21, 78. doi:10.1186/s13059-020-01989-2.

[97] Wu WY, Mohanraju P, Liao C, Adiego-Pérez B, Creutzburg SC, Makarova KS, et al. The miniature CRISPR-Cas12m effector binds DNA to block transcription. Mol. Cell 2022, 82, 4487-4502. doi:10.1016/j.molcel.2022.11.003.

[98] Chen W, Ma J, Wu Z, Wang Z, Zhang H, Fu W, et al. Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors. Mol. Cell 2023, 83, 2768-2780. doi:10.1016/j.molcel.2023.06.014.

[99] Awan MJ, Amin I, Mansoor S. CRISPR-Cas12c: a noncleaving DNA binder with minimal PAM requirement. Trends Biotechnol. 2022, 40, 1141-1143. doi:10.1016/j.tibtech.2022.07.005.

[100] Badon IW, Oh Y, Kim HJ, Lee SH. Recent application of CRISPR-Cas12 and OMEGA system for genome editing. Mol. Ther. 2024, 32, 32-43. doi:https://doi.org/10.1016/j.ymthe.2023.11.013.

[101] Kim DY, Lee JM, Moon SB, Chin HJ, Park S, Lim Y, et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat. Biotechnol. 2022, 40, 94-102. doi:10.1038/s41587-021-01009-z.

[102] Lim SR, Kim HJ, Lee SJ. Efficient CRISPR-Cas12f1-mediated multiplex bacterial genome editing via low-temperature recovery. J. Microbiol. Biotechnol. 2024, 34, 1522. doi:10.4014/jmb.2403.03033.

[103] Yan F, Wang J, Zhang S, Lu Z, Li S, Ji Z, et al. CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates. PLoS Pathogens. 2023, 19, e1010961. doi:10.1371/journal.ppat.1010961.

[104] Wang C, Xia Q, Zhang Q, Qu Y, Su S, Cheng JK, et al. CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells. Front. Cell Dev. Biol. 2022, 9, 719705. doi:10.3389/fcell.2021.719705.

[105] Jordan WT, Currie S, Schmitz RJ. Multiplex genome editing in Arabidopsis thaliana using Mb3Cas12a. Plant Direct. 2021, 5, e344. doi:10.1002/pld3.344.

[106] Hui F, Tang X, Li B, Alariqi M, Xu Z, Meng Q, et al. Robust CRISPR/Mb2Cas12a genome editing tools in cotton plants. Imeta 2024, 3, e209. doi:10.1002/imt2.209.

[107] Wang W, Li S, Yang J, Li J, Yan L, Zhang C, et al. Exploiting the efficient Exo: Cas12i3-5M fusions for robust single and multiplex gene editing in rice. J. Integr. Plant Biol. 2025, 67, 1246-1253. doi:10.1111/jipb.13850.

[108] Liu S, Sretenovic S, Fan T, Cheng Y, Li G, Qi A, et al. Hypercompact CRISPR-Cas12j2 (CasΦ) enables genome editing, gene activation, and epigenome editing in plants. Plant Commun. 2022, 3, 100453. doi:10.1016/j.xplc.2022.100453.

[109] Cui Y, Dong H, Tong B, Wang H, Chen X, Liu G, et al. A versatile Cas12k-based genetic engineering toolkit (C12KGET) for metabolic engineering in genetic manipulation-deprived strains. Nucleic Acids Res. 2022, 50, 8961-8973. doi:10.1093/nar/gkac655.

[110] Averina OA, Kuznetsova SA, Permyakov OA, Sergiev PV. Current knowledge of base editing and prime editing. Mol. Biol. 2024, 58, 571-587. doi:10.31857/S0026898424040013.

[111] Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage. Nature 2017, 551, 464-471. doi:10.1038/nature24644.

[112] Yan D, Ren B, Liu L, Yan F, Li S, Wang G, et al. High-efficiency and multiplex adenine base editing in plants using new TadA variants. Mol. Plant 2021, 14, 722-731. doi:https://doi.org/10.1016/j.molp.2021.02.007.

[113] Mishra R, Joshi RK, Zhao K. Genome editing in rice: recent advances, challenges, and future implications. Front. Plant Sci. 2018, 9, 1361. doi:10.3389/fpls.2018.01361.

[114] Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, et al. Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Front. Plant Sci. 2017, 8, 298. doi:10.3389/fpls.2017.00298.

[115] Xu Y, Lin Q, Li X, Wang F, Chen Z, Wang J, et al. Fine-tuning the amylose content of rice by precise base editing of the Wx gene. Plant Biotechnol. J. 2021, 19, 11-13. doi:10.1111/pbi.13433.

[116] Li J, Sun Y, Du J, Zhao Y, Xia L. Generation of targeted point mutations in rice by a modified CRISPR/Cas 9 system. Mol. Plant 2017, 10, 526-529. doi:10.1016/j.molp.2016.12.001.

[117] Liu X, Qin R, Li J, Liao S, Shan T, Xu R, et al. A CRISPR-Cas9-mediated domain-specific base-editing screen enables functional assessment of ACCase variants in rice. Plant Biotechnol. J. 2020, 18, 1845. doi:10.1111/pbi.13348.

[118] Tabassum J, Ahmad S, Hussain B, Mawia AM, Zeb A, Ju L. Applications and potential of genome-editing systems in rice improvement: current and future perspectives. Agronomy 2021, 11, 1359. doi:10.3390/agronomy11071359.

[119] Monsur MB, Shao G, Lv Y, Ahmad S, Wei X, Hu P, et al. Base editing: The ever expanding clustered regularly interspaced short palindromic repeats (CRISPR) tool kit for precise genome editing in plants. Genes 2020, 11, 466. doi:10.3390/genes11040466.

[120] Xie J, Huang X, Wang X, Gou S, Liang Y, Chen F, et al. ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems. BMC Biol. 2020, 18, 131. doi:10.1186/s12915-020-00866-5.

[121] Wu Y, Li Y, Liu Y, Xiu X, Liu J, Zhang L, et al. Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor. Nucleic Acids Res. 2024, 52, 4739-4755. doi:10.1093/nar/gkae228.

[122] Grünewald J, Zhou R, Lareau CA, Garcia SP, Iyer S, Miller BR, et al. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nat. Biotechnol. 2020, 38, 861-864. doi:10.1038/s41587-020-0535-y.

[123] Butt H, Rao GS, Sedeek K, Aman R, Kamel R, Mahfouz M. Engineering herbicide resistance via prime editing in rice. Plant Biotechnol. J. 2020, 18, 2370. doi:10.1111/pbi.13399.

[124] Gupta A, Liu B, Raza S, Chen QJ, Yang B. Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice. Plant Commun. 2024, 5. 100741. doi:https://doi.org/10.1016/j.xplc.2023.100741.

[125] Doman JL, Sousa AA, Randolph PB, Chen PJ, Liu DR. Designing and executing prime editing experiments in mammalian cells. Nat Protocols 2022, 17, 2431-2468. doi:10.1038/s41596-022-00724-4.

[126] Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 2022, 40, 402-410. doi:10.1038/s41587-021-01039-7.

[127] Awan MJ, Ali Z, Amin I, Mansoor S. Twin prime editor: seamless repair without damage. Trends Biotechnol. 2022, 40, 374-376. doi:10.1016/j.tibtech.2022.01.013.

[128] Wu K, Sánchez-Rivera FJ. It's prime time for multiplexed prime editing. Cell Genom. 2025, 5, 100852. doi:10.1016/j.xgen.2025.100852.

[129] Yuan Q, Gao X. Multiplex base-and prime-editing with drive-and-process CRISPR arrays. Nat. Commun. 2022, 13, 2771. doi:10.1038/s41467-022-30514-1.

[130] Zhao X, Yang L, Li P, Cheng Z, Jia Y, Luo L, et al. High-accuracy crRNA array assembly strategy for multiplex CRISPR. Mol. Ther. Nucleic Acids 2025, 36, 102428. doi:10.1016/j.omtn.2024.102428.

[131] Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, et al. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning. Biotechniques 2015, 58, 161-170. doi:10.2144/000114261.

[132] Breunig CT, Durovic T, Neuner AM, Baumann V, Wiesbeck MF, Köferle A, et al. One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR). PloS ONE 2018, 13, e0196015. doi:10.1371/journal.pone.0196015.

[133] Wu S, Kyaw H, Tong Z, Yang Y, Wang Z, Zhang L, et al. A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants. Crop J. 2024, 12, 569-582. doi:10.1016/j.cj.2024.01.010.

[134] Hsieh-Feng V, Yang Y. Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing. Abiotech 2020, 1, 123-134. doi:10.1007/s42994-019-00014-w.

[135] Dong F, Xie K, Chen Y, Yang Y, Mao Y. Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells. Biochem. Biophys. Res. Commun. 2017, 482, 889-895. doi:10.1016/j.bbrc.2016.11.129.

[136] Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc. Nat. Acad. Sci. USA 2015, 112, 3570-3575. doi:10.1073/pnas.1420294112.

[137] Jiang C, Geng L, Wang J, Liang Y, Guo X, Liu C, et al. Multiplexed gene engineering based on dCas9 and gRNA-tRNA array encoded on single transcript. Int. J. Mol. Sci. 2023, 24, 8535. doi:10.3390/ijms24108535.

[138] Gao Z, Herrera-Carrillo E, Berkhout B. Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA. RNA Biol. 2018, 15, 1458-1467. doi:10.1080/15476286.2018.1551703.

[139] Wang L, Han H. Strategies for improving the genome-editing efficiency of class 2 CRISPR/Cas system. Heliyon 2024, 10, e38588. doi:10.1016/J.HELIYON.2024.E38588.

[140] Xu L, Zhao L, Gao Y, Xu J, Han R. Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA. Nucleic acids Res. 2017, 45, e28. doi:10.1093/nar/gkw1048.

[141] Haurwitz RE, Sternberg SH, Doudna JA. Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA. The EMBO J. 2012, 31, 2824-2832. doi:10.1038/emboj.2012.107.

[142] Yan Q, Xu K, Xing J, Zhang T, Wang X, Wei Z, et al. Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure. Sci. Rep. 2016, 6, 38970. doi:10.1038/srep38970.

[143] Bevacqua RJ, Zhao W, Merheb E, Kim SH, Marson A, Gloyn AL, et al. Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein. iScience 2023, 27, 108693. doi:10.1016/J.ISCI.2023.108693.

[144] Bevacqua RJ, Zhao W, Merheb E, Kim SH, Marson A, Gloyn AL, et al. Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein. bioRxiv 2023, doi:10.1101/2023.09.16.558090.

[145] Nijkamp E, Ruffolo JA, Weinstein EN, Naik N, Madani A. Progen2: exploring the boundaries of protein language models. Cell Syst. 2023, 14, 968-978. doi:10.1016/j.cels.2023.10.002.

[146] McCarty NS, Graham AE, Studená L, Ledesma-Amaro R. Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat. Commun. 2020, 11, 1281. doi:10.1038/s41467-020-15053-x.

[147] Xun G, Zhu Z, Singh N, Lu J, Jain PK, Zhao H. Harnessing noncanonical crRNA for highly efficient genome editing. Nat. Commun. 2024, 15, 3823. doi:10.1038/s41467-024-48012-x.

[148] Bin Moon SU, Lee JM, Kang JG, Lee NE, Ha DI, Kim DY, et al. Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang. Nat. Commun. 2018, 9, 3651. doi:10.1038/s41467-018-06129-w.

[149] Liao C, Ttofali F, Slotkowski RA, Denny SR, Cecil TD, Leenay RT, et al. Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis. Nat. Commun. 2019, 10, 2948. doi:10.1038/s41467-019-10747-3.

[150] Erdoğan S. Integration of artificial intelligence and genome editing system for determining the treatment of genetic disorders. Balkan Med. J. 2024, 41, 419. doi:10.4274/balkanmedj.galenos.2024.2024-080824.

[151] Chen L, Liu G, Zhang T. Integrating machine learning and genome editing for crop improvement. Abiotech 2024, 5, 262-277. doi:10.1007/s42994-023-00133-5.

[152] Chia BS, Seah YF, Wang B, Shen K, Srivastava D, Chew WL. Engineering a New Generation of Gene Editors: Integrating Synthetic Biology and AI Innovations. ACS Synth. Biol. 2025, 14, 636-647. doi:10.1021/acssynbio.4c00686.

[153] Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 2014, 32, 1262-1267. doi:10.1038/nbt.3026.

[154] Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 2014, 42, W401-W407. doi:10.1093/nar/gku410.

[155] Li J, Wu P, Cao Z, Huang G, Lu Z, Yan J, et al. Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing. Cell Rep. 2024, 43, 113765. doi:10.1016/j.celrep.2024.113765.

[156] Alipanahi R, Safari L, Khanteymoori A. DTMP-prime: A deep transformer-based model for predicting prime editing efficiency and PegRNA activity. Mol. Ther. Nucleic Acids 2024, 35, 102370. doi:10.1016/j.omtn.2024.102370.

[157] Ruffolo JA, Nayfach S, Gallagher J, Bhatnagar A, Beazer J, Hussain R, et al. Design of highly functional genome editors by modelling CRISPR-Cas sequences. Nature 2025, 645, 518-525. doi:10.1038/s41586-025-09298-z.

[158] Kim MG, Go MJ, Kang SH, Jeong SH, Lim K. Revolutionizing CRISPR technology with artificial intelligence. Exp. Mol. Med. 2025, 57, 1419-1431. doi:10.1038/s12276-025-01462-9.

[159] Qu Y, Huang K, Yin M, Zhan K, Liu D, Yin D, et al. CRISPR-GPT for agentic automation of gene-editing experiments. Nat. Biomed. Eng. 2025, 1-4. doi:10.1038/s41551-025-01463-z.

[160] Du Y, Liu Y, Hu J, Peng X, Liu Z. CRISPR/Cas9 systems: Delivery technologies and biomedical applications. Asian J. Pharm. Sci. 2023, 18, 100854. doi:10.1016/j.ajps.2023.100854.

[161] Wang D, Tai PW, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug discov. 2019, 18, 358-378. doi:10.1038/s41573-019-0012-9.

[162] Lundstrom K. Viral vectors in gene therapy: where do we stand in 2023? Viruses 2023, 15, 698. doi:10.3390/v15030698.

[163] Sioson VA, Kim M, Joo J. Challenges in delivery systems for CRISPR-based genome editing and opportunities of nanomedicine. Biomed. Eng. Lett. 2021, 11, 217-233. doi:10.1007/s13534-021-00199-4.

[164] Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 2018, 25, 1234-1257. doi:10.1080/10717544.2018.1474964.

[165] Fang T, Cao X, Ibnat M, Chen G. Stimuli-responsive nanoformulations for CRISPR-Cas9 genome editing. J. Nanobiotechnol. 2022, 20, 354. doi:10.1186/s12951-022-01570-y.

[166] Zhang J, Zhou Y, Qiao J, Liu Y. Recent advances in spatiotemporal control of the CRISPR/Cas 9 system. Colloid. Surf. B Biointerf. 2025, 248, 114474. doi:https://doi.org/10.1016/j.colsurfb.2024.114474.

[167] Clarissa EM, Karmacharya M, Choi H, Kumar S, Cho YK. Nature Inspired Delivery Vehicles for CRISPR-Based Genome Editing. Small 2025, 2409353. doi:10.1002/smll.202409353.

[168] Lee H, Rho WY, Kim YH, Chang H, Jun BH. CRISPR-Cas9 gene therapy: non-viral delivery and stimuli-responsive nanoformulations. Molecules 2025, 30, 542. doi:10.3390/molecules30030542.

[169] Tashima T. Non-Invasive Delivery of CRISPR/Cas9 Ribonucleoproteins (Cas9 RNPs) into Cells via Nanoparticles for Membrane Transport. Pharmaceutics 2025, 17, 201. doi:10.3390/pharmaceutics17020201.

[170] Kaupbayeva B, Tsoy A, Safarova Y, Nurmagambetova A, Murata H, Matyjaszewski K, et al. Unlocking genome editing: Advances and obstacles in CRISPR/Cas delivery technologies. J. Funct. Biomat. 2024, 15, 324. doi:10.3390/jfb15110324.

[171] Shaikhutdinov IH, Ilyasov PV, Gribkova OV, Limareva LV. Non-viral systems for intracellular delivery of genome editing tools. Vavilov J. Genet.s Breeding 2024, 28, 239. doi:10.18699/vjgb-24-28.

[172] Alyami MZ, Alsaiari SK, Li Y, Qutub SS, Aleisa FA, Sougrat R, et al. Cell-type-specific CRISPR/Cas9 delivery by biomimetic metal organic frameworks. J. Am. Chem. Soc. 2020, 142, 1715-120. doi:10.1021/jacs.9b11638.

[173] Kangarshahi BM, Javazm AB, Naghib SM. CRISPR-integrated metal-organic frameworks for biosensing applications: Recent advances and future perspective. Sensing Bio-Sensing Res. 2025, 47, 100736. doi:https://doi.org/10.1016/j.sbsr.2025.100736.

[174] Yu J, Tang M, Zhou Z, Wei Z, Wan F, Hou S, et al. Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy. Acta Biomat. 2024, 178, 296-306. doi:10.1016/j.actbio.2024.02.030.

[175] Wang Q, Yu J, Kadungure T, Beyene J, Zhang H, Lu Q. ARMMs as a versatile platform for intracellular delivery of macromolecules. Nat. Commun. 2018, 9, 960. doi:10.1038/s41467-018-03390-x.

[176] Chen Z, Wang Q, Lu Q. Engineering ARMMs for improved intracellular delivery of CRISPR-Cas9. Extracell. Vesicle 2025, 5, 100082. doi:https://doi.org/10.1016/j.vesic.2025.100082.

[177] Halegua T, Risson V, Carras J, Rouyer M, Coudert L, Jacquier A, et al. Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles. Nat. Commun. 2025, 16, 397. doi:10.1038/s41467-024-55604-0.

[178] LaBauve AE, Saada EA, Jones IK, Mosesso R, Noureddine A, Techel J, et al. Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas 9 ribonucleoproteins. Sci. Rep. 2023, 13, 6873. doi:10.1038/s41598-023-33092-4.

[179] Graham JP, Castro JG, Werba LC, Amitrano J, Fardone L, Francis KP, et al. Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells. Adv. Funct. Mat. 2025, e01532. doi:10.1101/2024.09.23.614499.

[180] Borphukan B, Khatun M, Fartyal D, James D, Reddy MK. A Gemini Virus-Derived Autonomously Replicating System for HDR-Mediated Genome Editing of the EPSP Synthase Gene in Indica Rice. Plants 2025, 14, 477. doi:10.3390/plants14030477.

[181] Guo G, Li MJ, Lai JL, Du ZY, Liao QS. Development of tobacco rattle virus-based platform for dual heterologous gene expression and CRISPR/Cas reagent delivery. Plant Sci. 2022, 325, 111491. doi:10.1016/j.plantsci.2022.111491.

[182] Daròs JA, Pasin F, Merwaiss F. CRISPR-Cas-based plant genome engineering goes viral. Mol. Plant 2023, 16, 660-661. doi:10.1016/j.molp.2023.03.010.

[183] Hu J, Li S, Li Z, Li H, Song W, Zhao H, et al. A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize. Mol. Plant Pathol. 2019, 20, 1463-1474. doi:10.1111/mpp.12849.

[184] Mahmood MA, Naqvi RZ, Rahman SU, Amin I, Mansoor S. Plant virus-derived vectors for plant genome engineering. Viruses 2023, 15, 531. doi:10.3390/v15020531.

[185] Panda D, Karmakar S, Dash M, Tripathy SK, Das P, Banerjee S, et al. Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot. Abiotech 2024, 5, 151-168. doi:10.1007/s42994-024-00139-7.

[186] Vats S, Kumawat S, Brar J, Kaur S, Yadav K, Magar SG, et al. Opportunity and challenges for nanotechnology application for genome editing in plants. Plant Nano Biol. 2022, 1, 100001. doi:10.1016/j.plana.2022.100001.

[187] Bindal G, Misra CS, Singh M, Rangu SS, Pandey N, Rath D. High throughput CRISPR approaches: new solutions for challenging problems. TrAC Trends Analyt. Chem. 2025, 189, 118245. doi:10.1016/J.TRAC.2025.118245.