Research Progress on Optimization of CRISPR/Cas9 Gene Editing Technology Based on Chromatin Conformational Regulation
GUO Zi-Rong1, QIU Shuang1, CHEN Li-Qiang1, SU Guang-Hua1, ZHU Yi-Jing2,*, SU Xiao-Hu1,*
1 School of Life Sciences, Inner Mongolia University, Hohhot 010020, China; 2 Ulanqab Animal Husbandry Workstation of Inner Mongolia Autonomous Region, Ulanqab 012000, China
Abstract：The gene editing technology based on CRISPR/Cas9 is of great help to the basic biological research, disease treatment, and the improvement of animal and plant production traits. However, the editing efficiency varies greatly among loci. The reason for this is that chromatin conformation is one of the influencing factors. Based on this, researchers have conducted extensive research on the optimization of CRISPR/Cas9 gene editing technology based on chromatin conformational regulation. In order to promote the development of this field, this paper reviews the proteins and small molecules related to chromatin conformation regulation and their application progress in CRISPR/Cas9 gene editing optimization, aiming to provide some theoretical and technical reference for the research on gene editing efficiency improvement.
[1] 郭苇, 刘勤献, 汤明, 等. 2017. Sirtuins家族蛋白参与DNA损伤修复的研究进展[J]. 中国科学: 生命科学, 47(06): 595-605. (Guo W, Liu Q X, Tang M, et al.2017. Research progress on the involvement of Sirtuins family proteins in DNA damage repair[J]. Science in China: Life Sciences, 47(06): 595-605.) [2] 刘俊豪. 2020. 通过促进染色质开放水平提高Cas9基因组编辑效率的研究[D]. 硕士学位论文, 山东大学, 导师: 黄启来, pp. 40-42. ( Liu J H.2020. Improving the efficiency of Cas9 genome editing by promoting chromatin opening level[D]. Thesis for M. S., Shandong University, Supervisor: Huang Q L, pp. 40-42.) [3] 潘馨玥. 2020. CRISPR/Cas9系统介导的基因组编辑技术研究进展[J]. 凯里学院学报, 38(06): 74-78. (Pan X Y.2020. Research progress on CRISPR/Cas9 system-mediated genome editing technology[J]. Journal of Carey College, 38(06): 74-78.) [4] 万娟. 2016. CCDC152的功能研究[D]. 硕士学位论文, 陕西师范大学, 导师: 赵利军, 李发荣, pp. 29-48. (Wan J.2016. Functional study of CCDC152[D]. Thesis for M.S., Shaanxi Normal University, Supervisor: Zhao L J; Li F R, pp. 29-48.) [5] Allfrey V G, Faulkner R, Mirsky A E.1964. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis[J]. Proceedings of the National Academy of Sciences of the USA, 51(5): 786-794. [6] Aricthota S, Rana P P, Haldar D.2022. Histone acetylation dynamics in repair of DNA double-strand breaks[J]. Frontiers in Genetics, 13: 926577. [7] Bannister A J, Kouzarides T.2011. Regulation of chromatin by histone modifications[J]. Cell Research, 21(3): 381-395. [8] Bustin M.2001. Chromatin unfolding and activation by HMGN(*) chromosomal proteins[J]. Trends in Biochemical Sciences, 26(7): 431-437. [9] Cao F, Zwinderman M R H, van Merkerk R, et al.2019. Inhibitory selectivity among class I HDACs has a major impact on inflammatory gene expression in macrophages[J]. European Journal of Medicinal Chemistry, 177: 457-466. [10] Chen S, Chen D, Liu B, et al.2022. Modulating CRISPR/Cas9 genome-editing activity by small molecules[J]. Drug Discovery Today, 27(4): 951-966. [11] Chen X, Liu J, Janssen J M, et al.2017. The chromatin structure differentially impacts high-specificity CRISPR-Cas9 nuclease strategies[J]. Molecular Therapy-Nucleic Acids, 8: 558-563. [12] Chen X, Rinsma M, Janssen J M, et al.2016. Probing the impact of chromatin conformation on genome editing tools[J]. Nucleic Acids Research, 44(13): 6482-6492. [13] Cong L, Ran F A, Cox D, et al.2013. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 339(6121): 819-823. [14] De Sá Fernandes C, Novoszel P, Gastaldi T, et al.2024. The histone deacetylase HDAC1 controls dendritic cell development and anti-tumor immunity[J]. Cell Reports, 43(6): 114308. [15] Ding X, Seebeck T, Feng Y, et al.2019. Improving CRISPR-Cas9 genome editing efficiency by fusion with chromatin-modulating peptides[J]. CRISPR Journal, 2: 51-63. [16] Friskes A, Koob L, Krenning L, et al.2022. Double-strand break toxicity is chromatin context independent[J]. Nucleic Acids Research, 50(17): 9930-9947. [17] Ebrahimighaei R, McNeill M C, Smith S A, et al.2019. Elevated cyclic-AMP represses expression of exchange protein activated by cAMP (EPAC1) by inhibiting YAP-TEAD activity and HDAC-mediated histone deacetylation[J]. Biochimica et Biophysica Acta-Molecular Cell Research, 1866(10): 1634-1649. [18] Eghbalsaied S, Kues W A.2023. CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII[J]. Scientific Reports, 13(1): 2690. [19] Gaspar-Maia A, Alajem A, Polesso F, et al.2009. Chd1 regulates open chromatin and pluripotency of embryonic stem cells[J]. Nature, 460(7257): 863-868. [20] González-Romero R, Eirín-López J M, Ausió J.2015. Evolution of high mobility group nucleosome-binding proteins and its implications for vertebrate chromatin specialization[J]. Molecular Biology and Evolution, 32(1): 121-131. [21] Haberland M, Montgomery R L, Olson E N.2009. The many roles of histone deacetylases in development and physiology: Implications for disease and therapy[J]. Nature Reviews Genetics, 10(1): 32-42. [22] Harshman S W, Young N L, Parthun M R, et al.2013. H1 histones: Current perspectives and challenges[J]. Nucleic Acids Research, 41(21): 9593-9609. [23] Hinz J M, Laughery M F, Wyrick J J.2015. Nucleosomes inhibit Cas9 endonuclease activity in vitro[J]. Biochemistry, 54(48): 7063-7066. [24] Hinz J M, Laughery M F, Wyrick J J.2016. Nucleosomes selectively inhibit Cas9 off-target activity at a site located at the nucleosome edge[J]. Journal of Biological Chemistry, 291(48): 24851-24856. [25] Isaac R S, Jiang F, Doudna J A, et al.2016. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function[J]. Elife, 5: e13450. [26] Jensen K T, Fløe L, Petersen T S, et al.2017. Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency[J]. FEBS Letters, 591(13): 1892-1901. [27] Klass J, Murphy F V T, Fouts S, et al.2003. The role of intercalating residues in chromosomal high-mobility-group protein DNA binding, bending and specificity[J]. Nucleic Acids Research, 31(11): 2852-2864. [28] Kouzarides T.2007. Chromatin modifications and their function[J]. Cell, 128(4): 693-705. [29] Kugler J E, Deng T, Bustin M.2012. The HMGN family of chromatin-binding proteins: Dynamic modulators of epigenetic processes[J]. Biochimica et Biophysica Acta, 1819(7): 652-656. [30] Kuscu C, Arslan S, Singh R, et al.2014. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease[J]. Nature Biotechnology, 32(7): 677-683. [31] Li G, Yang X, Luo X, et al.2023. Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair[J]. Cell Bioscience, 13(1): 215. [32] Li G, Zhang X, Wang H, et al.2020. Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors[J]. International Journal of Biochemistry and Cell Biology, 125: 105790. [33] Li Y, Gong H, Wang P, et al.2021. The emerging role of ISWI chromatin remodeling complexes in cancer[J]. Journal of Experimental & Clinical Cancer Research, 40(1): 346. [34] Lin S, Staahl B T, Alla R K, et al.2014. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery[J]. Elife, 3: 04766. [35] Lin Y, Qiu T, Wei G, et al.2022. Role of histone post-translational modifications in inflammatory diseases[J]. Frontiers in Immunology, 13: 852272. [36] Liu B, Chen S, Rose A, et al.2020. Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing[J]. Nucleic Acids Research, 48(2): 517-532. [37] Maruyama T, Dougan S K, Truttmann M C, et al.2015. Increasing the efficiency of precise genome editing with CRISPR/Cas9 by inhibition of nonhomologous end joining[J]. Nature Biotechnology, 33(5): 538-542. [38] Pachano B, Farhat D C, Shahinas M, et al.2025. An ISWI-related chromatin remodeller regulates stage-specific gene expression in Toxoplasma gondii[J]. Nature Microbiology, 10(5): 1156-1170. [39] Pan Z, Oh J, Huang L, et al.2020. The combination of forskolin and VPA increases gene expression efficiency to the hypoxia/neuron-specific system[J]. Annals of Translational Medicine, 8(15): 933. [40] Persson J, Ekwall K.2010. Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation[J]. Experimental Cell Research, 316(8): 1316-1323. [41] Probst A V, Almouzni G.2008. Pericentric heterochromatin: Dynamic organization during early development in mammals[J]. Differentiation, 76(1): 15-23. [42] Rahmani B, Kheirandish M H, Ghanbari S, et al.2023. Targeting DNA repair pathways with B02 and nocodazole small molecules to improve CRIS-PITCH mediated cassette integration in CHO-K1 cells[J]. Scientific Reports, 13(1): 3116. [43] Schubert T, Pusch M C, Diermeier S, et al.2012. DF31 protein and snoRNAs maintain accessible higher-order structures of chromatin[J]. Molecular Cell, 48(3): 434-444. [44] Shin H R, See J E, Kweon J, et al.2021. Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing[J]. Nucleic Acids Research, 49(4): 2390-2399. [45] Shin J, Oh J W.2020. Development of CRISPR/Cas9 system for targeted DNA modifications and recent improvements in modification efficiency and specificity[J]. BMB Reports, 53(7): 341-348. [46] Singharajkomron N, Seephan S, Iksen I, et al.2024. CAMSAP3-mediated regulation of hmgb1 acetylation and subcellular localization in lung cancer cells: Implications for cell death modulation[J]. Biochimica et Biophysica Acta - General Subjects, 1868(6): 130614. [47] Sivaraj D, Green M M, Gasparetto C.2017. Panobinostat for the management of multiple myeloma[J]. Future Oncology, 13(6): 477-488. [48] Smith D K, Yang J, Liu M L, et al.2016. Small molecules modulate chromatin accessibility to promote neurog2-mediated fibroblast-to-neuron reprogramming[J]. Stem Cell Reports, 7(5): 955-969. [49] Sun X J, Man N, Tan Y, et al.2015. The role of histone acetyltransferases in normal and malignant hematopoiesis[J]. Frontiers in Oncology, 5: 108. [50] Takata M, Sasaki M S, Sonoda E, et al.1998. Homologous recombination and non-homologous end-joining pathways of dna double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells[J]. The EMBO Journal, 17(18): 5497-5508. [51] Tate C R, Rhodes L V, Segar H C, et al.2012. Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat[J]. Breast Cancer Research, 14(3): R79. [52] Thomas J O, Stott K.2012. H1 and HMGB1: Modulators of chromatin structure[J]. Biochemical Society Transactions, 40(2): 341-346. [53] Verkuijl S A, Rots M G.2019. The influence of eukaryotic chromatin state on CRISPR/Cas9 editing efficiencies[J]. Current Opinion in Biotechnology, 55: 68-73. [54] Wang S, Huang J, Lyu H, et al.2013. Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells[J]. Cell Death & Disease, 4(3): e556. [55] Wang X, Niu Y, Zhou J, et al.2016. Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep[J]. Scientific Reports, 6(1): 10064. [56] Wu X, Scott D A, Kriz A J, et al.2014. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells[J]. Nature Biotechnology, 32(7): 670-676. [57] Yang C, Ma Z, Wang K, et al.2023. HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing[J]. Nature Communications, 14(1): 2430. [58] Yao X, Zhang M, Wang X, et al.2018. Tild-CRISPR allows for efficient and precise gene knockin in mouse and human cells[J]. Developmental Cell, 45(4): 526-536.e525. [59] Yarrington R M, Verma S, Schwartz S, et al.2018. Nucleosomes inhibit target cleavage by CRISPR/Cas9 in vivo[J]. Proceedings of the National Academy of Sciences of the USA, 115(38): 9351-9358. [60] Yoon S, Eom G H.2016. HDAC and HDAC inhibitor: From cancer to Cardiovascular diseases[J]. Chonnam Medical Journal, 52(1): 1-11. [61] Yu W, Lescale C, Babin L, et al.2020. Repair of G1 induced dna double-strand breaks in S-G2/M by alternative NHE[J][J]. Nature Communications, 11(1): 5239. [62] Zhang B, Luo D, Li Y, et al.2021a. Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1[J]. Nature Communications, 12(1): 3476. [63] Zhang J P, Li X L, Li G H, et al.2017. Efficient precise knockin with a double cut hdr donor after CRISPR/Cas9-mediated double-stranded DNA cleavage[J]. Genome Biology, 18(1): 35. [64] Zhang J P, Yang Z X, Zhang F, et al.2021b. Hdac inhibitors improve CRISPR-mediated HDR editing efficiency in IPSCS[J]. Science China Life Sciences, 64(9): 1449-1462. [65] Zhu Y.2022. Advances in CRISPR/Cas9[J]. Biomedical Research International, 2022: 9978571.
