Construction and Efficiency Evaluation of the Cas12a-mediated Site-specific Knock-in System in Pig Cells

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

Abstract

Abstract:

Objective This study 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

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, 2026, 42(8): 72-80.

Figures/Tables 4

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

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

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 editing sites

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

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