Thomas Holly R, Yoder Bradley K, Alexander Matthew S, Parant John M
Department of Cell, Developmental and Integrative Biology, Heersink School of Medicine, University of Alabama at Birmingham, AL, USA.
Department of Pediatrics, Division of Neurology at University of Alabama at Birmingham Heersink School of Medicine and Children's of Alabama, AL, USA.
bioRxiv. 2025 May 21:2025.05.18.654701. doi: 10.1101/2025.05.18.654701.
Animal models of human diseases are an essential component of understanding disease pathogenesis and serve as preclinical models for therapeutic evaluation. Recently human patient genome sequencing has defined unique patient variants that result in disease states with different phenotypes than those observed with null alleles. The UAB Center for Precision Animal Modeling (CPAM) serves to analyze patient variant pathogenicity and disease mechanisms through the generation of animal models. We have optimized a zebrafish gene editing platform to successfully generate 11 patient variants (first round: NF1 R1276Q, NF1 G484R, VMA21 G55V, SPOP D144N, SGO1 K23E, Pex10 H310D, and FKRP C318Y; second round: NF1 R681*, NF1 M992del, P53 R175H, and PKD2 L656W) and 1 research allele ( K120R). We used CRISPR/Cas9 guide directed cleavage along with single-stranded oligodeoxynucleotide (ssODNs) repair templates to generate these models. We evaluated multiple oligo orientations and sizes, but did not find a unified consensus orientation or size that significantly impacted efficiency, emphasizing the need to empirically evaluate multiple variations for the best homology directed repair (HDR) rate. We determined PCR amplicon Next Generation Sequencing (NGS) evaluation of HDR efficiency at the F0 embryo level is best for determining the ideal guide and oligo combination. Further NGS evaluation of DNA from progeny from F0s (germline derived), not F0 biopsy DNA, is essential to identify germline transmitting founders. Surprisingly we find that most founders exhibit a effect in the germ line but not in the somatic tissue. We found NGS superior to using ICE (Inference of CRISPR Edits) for determining HDR frequency. When applicable, allelic-specific PCR or allelic specific restriction digestion can be used to genotype mutation carrying F1 generation animals, however we demonstrated that false positives occur. Further, we successfully used high resolution melting curve analysis (HRMA) to differentiate and identify F1 animals with patient variants.
人类疾病的动物模型是理解疾病发病机制的重要组成部分，并且作为治疗评估的临床前模型。最近，人类患者基因组测序确定了独特的患者变体，这些变体导致的疾病状态具有与无效等位基因所观察到的不同的表型。阿拉巴马大学伯明翰分校精准动物建模中心（CPAM）致力于通过生成动物模型来分析患者变体的致病性和疾病机制。我们优化了一个斑马鱼基因编辑平台，成功生成了11种患者变体（第一轮：NF1 R1276Q、NF1 G484R、VMA21 G55V、SPOP D144N、SGO1 K23E、Pex10 H310D和FKRP C318Y；第二轮：NF1 R681*、NF1 M992del、P53 R175H和PKD2 L656W）以及1个研究等位基因（K120R）。我们使用CRISPR/Cas9引导的切割以及单链寡脱氧核苷酸（ssODN）修复模板来生成这些模型。我们评估了多种寡核苷酸方向和大小，但未找到对效率有显著影响的统一一致的方向或大小，这强调了需要通过实验评估多种变体以获得最佳的同源定向修复（HDR）率。我们确定在F0胚胎水平对HDR效率进行PCR扩增子下一代测序（NGS）评估最适合确定理想的引导序列和寡核苷酸组合。对F0后代（种系来源）而非F0活检DNA的DNA进行进一步的NGS评估对于鉴定种系传递奠基者至关重要。令人惊讶的是，我们发现大多数奠基者在种系中表现出效应，但在体细胞组织中未表现出效应。我们发现NGS在确定HDR频率方面优于使用ICE（CRISPR编辑推断）。在适用时，等位基因特异性PCR或等位基因特异性限制性消化可用于对携带突变的F1代动物进行基因分型，然而我们证明会出现假阳性。此外，我们成功地使用高分辨率熔解曲线分析（HRMA）来区分和鉴定具有患者变体的F1动物。
