Browsing by Author "Pan, Yidan"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing
    (AAAS, 2022) Park, So Hyun; Cao, Mingming; Pan, Yidan; Davis, Timothy H.; Saxena, Lavanya; Deshmukh, Harshavardhan; Fu, Yilei; Treangen, Todd; Sheehan, Vivien A.; Bao, Gang
    Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.
  • Thumbnail Image
    Item
    Fine-mapping within eQTL credible intervals by expression CROP-seq
    (Oxford University Press, 2020) Pan, Yidan; Tian, Ruoyu; Lee, Ciaran; Bao, Gang; Gibson, Greg; Bioengineering
    The majority of genome-wide association study (GWAS)-identified SNPs are located in noncoding regions of genes and are likely to influence disease risk and phenotypes by affecting gene expression. Since credible intervals responsible for genome-wide associations typically consist of ≥100 variants with similar statistical support, experimental methods are needed to fine map causal variants. We report here a moderate-throughput approach to identifying regulatory GWAS variants, expression CROP-seq, which consists of multiplex CRISPR-Cas9 genome editing combined with single-cell RNAseq to measure perturbation in transcript abundance. Mutations were induced in the HL60/S4 myeloid cell line nearby 57 SNPs in three genes, two of which, rs2251039 and rs35675666, significantly altered CISD1 and PARK7 expression, respectively, with strong replication and validation in single-cell clones. The sites overlap with chromatin accessibility peaks and define causal variants for inflammatory bowel disease at the two loci. This relatively inexpensive approach should be scalable for broad surveys and is also implementable for the fine mapping of individual genes.
  • Thumbnail Image
    Item
    Profiling Genome Editing Outcomes for Biological Studies and Disease Treatment
    (2020-04-23) Pan, Yidan; Bao, Gang
    CRISPR/Cas9 systems are designed to make site- and sequence-specific alterations to the genomes of a wide variety of organisms by targeting with user-defined guide RNAs (gRNAs) and Cas9 nucleases. Their high degree of efficiency and ease of construction in performing genome editing have led to a revolution in life sciences and medicine. However, challenges in quantifying genome editing outcomes need to be addressed. In this work, we first utilized CRISPR/Cas9 mediated genome editing to introduce small indel (insertion and deletion) mutations in the nearby regions of pre-selected single nucleotide polymorphisms (SNPs) and evaluated the potential of this approach for fine mapping causal variants to identify expression quantitative trait loci (eQTL) associated with inflammatory bowel disease. Our approach, expression CROP-seq, consists of multiplexed CRISPR/Cas9 genome editing followed by single-cell RNA-seq to measure perturbations in transcript abundance. We found that two SNPs out of 67 total target loci significantly altered the expressions of CISD1 and PARK7 genes, respectively. The expression CROP-seq approach is relatively inexpensive and has the capability of large scale fine-mapping. We further addressed two unmet needs that are critical for therapeutic genome editing: the profiling of on-target mutagenesis patterns and the analysis of genome-wide off-target effects. CRISPR/Cas9 induced DNA double-strand breaks result in large deletions and complex genomic rearrangements, which cannot be detected by short-read Next Generation Sequencing (NGS). Therefore, we developed long-range PCR based assays to profile on-target CRISPR/Cas9 mutagenesis patterns. Our results confirmed that the current gold standard of short-range PCR based assessment is unable to detect alleles containing large deletions. We also found that the profiles of large deletions are gRNA- and locus-specific. We further demonstrated that individual-specific genome variants could change the target-site profiling of gRNA designs. Our evaluation of current off-target prediction algorithms demonstrated the lack of capability to identify novel individual-specific off-target sites created by SNPs. To address this issue, we developed a bioinformatics tool that accommodates personal genomes for off-target identification to facilitate personalized therapeutic genome editing. In summary, we have developed quantitative tools for profiling genome editing outcomes that can be used widely for biological studies and disease treatment.
  • Thumbnail Image
    Item
    The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells
    (Springer Nature, 2021) Sharma, Rajiv; Dever, Daniel P.; Lee, Ciaran M.; Azizi, Armon; Pan, Yidan; Camarena, Joab; Köhnke, Thomas; Bao, Gang; Porteus, Matthew H.; Majeti, Ravindra; Bioengineering
    Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.