Cas12a介导的猪细胞定点插入体系构建与效率评估

doi:10.13560/j.cnki.biotech.bull.1985.2025-1408

摘要/Abstract

摘要：

目的系统评估CRISPR-Cas12a（Cpf1）系统在猪细胞中介导基因定点插入（knock-in）的潜能，并为其在猪基因组精准编辑中的应用提供实验依据与理论支撑。方法构建基于Acidaminococcus sp. Cas12a（AsCas12a）与Lachnospiraceae bacterium Cas12a（LbCas12a）的表达载体，并针对猪基因组安全位点Rosa26与AAVS1设计特异性crRNA，旨在比较两种Cas12a变体的切割效能。结果在AAVS1位点，AsCas12a的两条crRNA编辑效率分别为2.6%与1.2%，而LbCas12a对应的效率分别为6.5%与2.5%；在Rosa26位点，AsCas12a与LbCas12a的编辑效率分别为5.0%与12.7%，LbCas12a在猪源细胞中展现出相对较高的编辑活性。在此基础上，本研究构建了基于同源重组（HDR）、同源介导末端连接（HMEJ）、微同源介导末端连接（MMEJ）及非同源末端连接（NHEJ）4种策略的敲入载体，并评估了其整合效率。结果表明，上述4种修复途径均可介导目标基因在Rosa26和AAVS1位点的定点整合，其中HDR效率为35.0%、HMEJ（25.0%）、NHEJ与MMEJ均为15.0%。结论基于Cas12a基因编辑系统，在猪细胞中成功构建了4种定点插入策略，并对其效率进行了系统评估，该研究成果为拓展Cas12a系统在猪基因工程领域的应用提供了重要的实验依据和参考资料。

关键词: Cas12a, 猪, 定点插入, 基因编辑

Abstract:

Objective This study is aimed to systematically evaluate the potential of the CRISPR-Cas12a (Cpf1) system mediating site-specific gene knock-in in porcine cells, thereby providing an experimental basis and theoretical reference for its application in precise porcine genome engineering. Method Expression vectors for Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a) were constructed. Specific crRNAs were designed to target the well-characterized porcine "safe harbor" loci, Rosa26 and AAVS1, to compare the cleavage efficiencies of the two Cas12a variants. Result At the AAVS1 locus, the editing efficiencies of two crRNAs for AsCas12a were 2.6% and 1.2%, respectively, whereas those for LbCas12a were 6.5% and 2.5%. At the Rosa26 locus, the editing efficiencies for AsCas12a and LbCas12a were 5.0% and 12.7%, respectively, suggesting that LbCas12a possessed relatively higher editing activity in porcine cells. Based on these findings, knock-in donors were developed to utilize four distinct DNA repair pathways: Homology-directed repair (HDR), homology-mediated end joining (HMEJ), microhomology-mediated end joining (MMEJ), and non-homologous end joining (NHEJ), and their integration efficiencies were evaluated. The results revealed that 4 repair pathways all mediated the target genes to have site-targeted integration at both Rosa26 and AAVS1 loci. Specifically, the efficiency by HDR achieved 35.0%, 25.0% by HMEJ, and 15.0% by both NHEJ and MMEJ. Conclusion Four site-specific integration strategies are successfully developed in porcine cells using the Cas12a gene-editing system, followed by a systematic evaluation of their efficiencies. These findings provide essential experimental evidence and references for expanding the application of the Cas12a system in porcine genetic engineering.

Key words: Cas12a, pig, site-specific gene insertion, gene editing

曾亚丹, 毕佳俊, 李兴龙, 常悦, 角德灵, 魏红江, 赵恒. Cas12a介导的猪细胞定点插入体系构建与效率评估[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1408.

ZENG Ya-dan, BI Jia-jun, LI Xing-long, CHANG Yue, JIAO De-ling, WEI Hong-jiang, ZHAO Heng. Construction and Efficiency Evaluation of the Cas12a-mediated Site-specific Knock-in System in Pig Cells[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1408.

图/表 4

图1 AsCas12a和LbCas12a载体的构建和功能验证A：构建的AsCas12a、LbCas12a载体图谱；B：AsCas12a、LbCas12a载体转染PIEC后48 h在荧光显微镜下观察到细胞呈现绿色荧光；C：转染细胞的流式分析结果。AsCas12a和LbCas12a质粒转染细胞后，分别有74.7%和18.8%的细胞呈现绿色荧光；D：AAVS1和Rosa26基因靶位点设计示意图；E：AsCas12a和LbCas12a靶向AAVS1位点的细胞荧光图；F：细胞流式分析

Fig. 1 Construction and functional validation of AsCas12a and LbCas12a vectorsA: Profile of the constructed AsCas12a and LbCas12a vectors. B: Green fluorescence was observed in the cells at 48 h after transfecting PIEC cells with AsCas12a and LbCas12a vectors under a fluorescence microscope. C: Flow cytometry analysis result of transfected cells. The 74.7% and 18.8% of the cells, respectively, showed green fluorescence, after transfection with AsCas12a and LbCas12a plasmids. D: Schematic diagram of the design of gene targeting at AAVS1 and Rosa26 loci. E: Fluorescence images of cells targeting the AAVS1 locus with AsCas12a and LbCas12a. F: Flow cytometry analysis of the cells

图2 AsCas12a和LbCas12a在AAVS1和Rosa26基因靶位点编辑效率分析A：LbCas12a AAVS1 crRNA1、LbCas12a AAVS1 crRNA2和LbCas12a Rosa26 crRNA转染细胞基因组的PCR结果图（M：Marker 2 000，WT： wild type）。B：AsCas12a AAVS1 crRNA1、AsCas12a AAVS1 crRNA2和AsCas12a Rosa26crRNA转染细胞基因组的PCR结果图（M：Marker 2 000，WT： wild type）。C：PCR测序结果的峰值图。D：TIDE分析LbCas12a AAVS1 crRNA1、LbCas12a AAVS1 crRNA2 PCR测序结果。E：AsCas12a AAVS1 crRNA1、AsCas12a AAVS1 crRNA2和AsCas12a Rosa26 crRNA PCR扩增产物的TIDE分析

Fig. 2 Analysis of editing efficiency of AsCas12a and LbCas12a at AAVS1 and Rosa26 lociA: PCR results of the genomic DNA from the cells transfected with LbCas12a AAVS1 crRNA1, LbCas12a AAVS1 crRNA2, and LbCas12a Rosa26 crRNA (M: Marker 2 000. WT: wild type). B: PCR results of the genomic DNA from the cells transfected with AsCas12a AAVS1 crRNA1, AsCas12a AAVS1 crRNA2, and AsCas12a Rosa26 crRNA (M: Marker 2 000, WT: wild type). C: Peak plot of the PCR sequencing results. D: TIDE analysis of the sequencing results from LbCas12a AAVS1 crRNA1 and LbCas12a AAVS1 crRNA2 PCR. E: TIDE analysis of the PCR amplification products of AsCas12a AAVS1 crRNA1, AsCas12a AAVS1 crRNA2, and AsCas12a Rosa26 crRNA

图3 通过FastNGS分析LbCas12a系统在猪PIEC细胞中的编辑准确性和突变性A：LbCas12a介导的AAVS1基因crRNA1靶位点PCR产物的高通量测序分析，共获得2 176条测序reads。B：LbCas12a介导的Rosa26基因crRNA2靶位点PCR产物的高通量测序分析，1 997条reads。加粗字体表示碱基突变；红色方框表示碱基插入；短横线（-）表示碱基缺失；虚线指示预测的编辑位点

Fig. 3 Editing Accuracy and Mutagenesis of the LbCas12a System in Pig PIEC Cells Analyzed by FastNGSA: High-throughput sequencing analysis of the PCR products from the AAVS1 gene crRNA1 target site mediated by LbCas12a, with a total of 2 176 sequencing reads obtained. B: High-throughput sequencing analysis of the PCR products from the Rosa26 gene crRNA2 target site mediated by LbCas12a, with 1 997 sequencing reads obtained. Bold characters indicate base mutations; red boxes indicate nucleotide insertions; hyphens (-) indicate deletions; and dotted lines denote predicted cleavage/editing sites

图4 LbCas12a介导AAVS1和Rosa26基因靶位点在4种不同敲入路径的效率评估A：Cas12a介导HR、HMEJ、MMEJ和NHEJ四种基因敲入路径的机制示意图。B：Cas12a介导4种基因敲入路径的细胞筛选流程图。C：Cas12a介导HR、NHEJ、MMEJ、HMEJ四种敲入途径在AAVS1和Rosa26位点定点整合的单细胞阳性个数统计。D：Cas12a介导HR、NHEJ、MMEJ、HMEJ四种敲入途径在AAVS1和Rosa26位点定点整合的单细胞编辑效率结果。E：LbCas12a介导AAVS1基因crRNA1靶位点和Rosa26基因crRNA靶位点质粒转染PIEC细胞48小时后的荧光结果。F：LbCas12a介导AAVS1基因crRNA1靶位点和Rosa26基因crRNA靶位点质粒转染PIEC细胞48小时后的流式分选检测结果。G：LbCas12a介导AAVS1和Rosa26基因crRNA1靶位点的NHEJ、HMEJ、HDR和MMEJ修复途径PCR鉴定结果。图中的1-10和11-20为两次转染后每个单细胞经独立PCR扩增的凝胶图，WT：野生型DNA组

Fig. 4 Efficiency evaluation of the four different gene knock-in pathways at AAVS1 and Rosa26 gene loci mediated by LbCas12aA: Schematic diagram of the mechanisms of Cas12a-mediated HR, NHEJ, MMEJ, and HMEJ gene knock-in pathways. B: Cell screening flowchart for the four Cas12a-mediated gene knock-in pathways. C: Statistics of the number of positive single cells for targeted integration via Cas12a-mediated HR, NHEJ, MMEJ, and HMEJ pathways at the AAVS1 and Rosa26 loci. D: Targeted integration efficiency results for Cas12a-mediated HR, NHEJ, MMEJ, and HMEJ repair pathways at the AAVS1 and Rosa26 loci in single cells. E: Fluorescence results at 48 h after plasmid transfection targeting the AAVS1 crRNA1 locus and Rosa26 crRNA locus in PIEC cells mediated by LbCas12a. F: Flow cytometry results at 48 h after plasmid transfection targeting the AAVS1 crRNA1 locus and Rosa26 crRNA locus in PIEC cells mediated by LbCas12a. G: PCR-identified results of NHEJ, HMEJ, HDR, and MMEJ repair pathways mediated by LbCas12a at the crRNA1 target sites of AAVS1 and Rosa26. The gel images (lane 1-10 and 11-20) indicate independent PCR amplifications from single cells across two separate transfections. WT: Wild-type DNA control

参考文献 30

[1]	Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system [J]. Cell, 2015, 163(3): 759-771.
[2]	王炀坤, 纪世新, 苏莹莹, 等. 基因编辑工具CRISPR-Cas9与CRISPR-Cas12a的比较与应用 [J]. 长春师范大学学报, 2023, 42(12): 107-112.
	Wang YK, Ji SX, Su YY, et al. The diversity and applications of CRISPR-Cas9 and CRISPR-Cas12a [J]. J Changchun Norm Univ, 2023, 42(12): 107-112.
[3]	Fonfara I, Richter H, Bratovič M, et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA [J]. Nature, 2016, 532(7600): 517-521.
[4]	Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases [J]. Nat Biotechnol, 2013, 31(9): 827-832.
[5]	Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9 [J]. Science, 2014, 346(6213): 1258096.
[6]	Li JF, Norville JE, Aach J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 [J]. Nat Biotechnol, 2013, 31(8): 688-691.
[7]	Ran FA, Hsu PD, Wright J, et al. Genome engineering using the CRISPR-Cas9 system [J]. Nat Protoc, 2013, 8(11): 2281-2308.
[8]	Mali P, Yang LH, Esvelt KM, et al. RNA-guided human genome engineering via Cas9 [J]. Science, 2013, 339(6121): 823-826.
[9]	Lin S, Staahl BT, Alla RK, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery [J]. eLife, 2014, 3: e04766.
[10]	Schubert MS, Thommandru B, Woodley J, et al. Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair [J]. Sci Rep, 2021, 11: 19482.
[11]	Maruyama T, Dougan SK, Truttmann MC, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining [J]. Nat Biotechnol, 2015, 33(5): 538-542.
[12]	Tan JT, Zhao YC, Wang B, et al. Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining [J]. Plant Biotechnol J, 2020, 18(11): 2161-2163.
[13]	Yao X, Wang X, Hu XD, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9 [J]. Cell Res, 2017, 27(6): 801-814.
[14]	Huang J, Rowe D, Subedi P, et al. CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae [J]. Nat Commun, 2022, 13: 7168.
[15]	Cao L, Dong Y, Sun KW, et al. Experimental animal models for moyamoya disease: a species-oriented scoping review [J]. Front Surg, 2022, 9: 929871.
[16]	Gawel K, Langlois M, Martins T, et al. Seizing the moment: Zebrafish epilepsy models [J]. Neurosci Biobehav Rev, 2020, 116: 1-20.
[17]	Ely ZA, Mathey-Andrews N, Naranjo S, et al. A prime editor mouse to model a broad spectrum of somatic mutations in vivo [J]. Nat Biotechnol, 2024, 42(3): 424-436.
[18]	Lunney JK, Van Goor A, Walker KE, et al. Importance of the pig as a human biomedical model [J]. Sci Transl Med, 2021, 13(621): eabd5758.
[19]	Hou NP, Du XG, Wu S. Advances in pig models of human diseases [J]. Anim Models Exp Med, 2022, 5(2): 141-152.
[20]	Huang WZ, Xiong TL, Zhao YT, et al. Computational prediction and experimental validation identify functionally conserved lncRNAs from zebrafish to human [J]. Nat Genet, 2024, 56(1): 124-135.
[21]	董战旗, 秦琪, 张新铃, 等. 家蚕CRISPR/Cpf1基因编辑系统建立 [J]. 生物工程学报, 2021, 37(12): 4342-4350.
	Dong ZQ, Qin Q, Zhang XL, et al. Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori [J]. Chin J Biotechnol, 2021, 37(12): 4342-4350.
[22]	Tang KY, Zhou LQ, Tian XL, et al. Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering [J]. Nat Biomed Eng, 2025, 9(8): 1290-1308.
[23]	Sun RR, Zhao YQ, Wang WJ, et al. Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage [J]. Chem Sci, 2023, 14(14): 3839-3851.
[24]	Bernabé-Orts JM, Casas-Rodrigo I, Minguet EG, et al. Assessment of Cas12a-mediated gene editing efficiency in plants [J]. Plant Biotechnol J, 2019, 17(10): 1971-1984.
[25]	Zhang LY, Li G, Zhang YX, et al. Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants [J]. Genome Biol, 2023, 24: 102.
[26]	Anders C, Niewoehner O, Duerst A, et al. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease [J]. Nature, 2014, 513(7519): 569-573.
[27]	Li P, Zhang LJ, Li ZF, et al. Cas12a mediates efficient and precise endogenous gene tagging via MITI microhomology-dependent targeted integrations [J]. Cell Mol Life Sci, 2020, 77(19): 3875-3884.
[28]	Allen D, Knop O, Itkowitz B, et al. CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications [J]. Nat Commun, 2023, 14: 6771.
[29]	Van Vu T, Thi Hai Doan D, Kim J, et al. CRISPR/Cas-based precision genome editing via microhomology-mediated end joining [J]. Plant Biotechnol J, 2021, 19(2): 230-239.
[30]	Naqvi MM, Lee L, Montaguth OET, et al. CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage [J]. Nat Chem Biol, 2022, 18(9): 1014-1022.