pubmed.ncbi.nlm.nih.gov

Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells - PubMed

  • ️Sat Jan 01 2022

Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells

Tomoyuki Ohno et al. Nat Commun. 2022.

Abstract

Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we establish a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ~100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a standard platform with which to pursue the design of noncoding regions for genome writing in human cells.

© 2022. The Author(s).

PubMed Disclaimer

Conflict of interest statement

Patent application has been filed for the technology described in this manuscript: patent applicant: - Logomix, Inc. - Tokyo Institute of Technology name of inventor(s): - T.O. and Y.A. are named as the inventors of the patent for technologies related to UKiS. application number: - JP2020-068266 - WO2021/206054 A1 status of application: - In process of entering into the National Phase specific aspect of manuscript covered in patent application - The methodology of UKiS Y.A. is a co-founder and the chief scientific officer of Logomix, Inc. and T.O. is an employee of Logomix, Inc. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overview of UKiS.

a The two-step process of UKiS. First step: co-transfection of the expression plasmid for Cas9 protein and the gene-specific gRNA and the two UKiS donor plasmids, followed by puromycin and blasticidin selection, to collect cells that had undergone biallelic replacement of the target locus with UKiS marker sequences. Second step: co-transfection of the expression plasmid for Cas9 and the Off-Target Less gRNA (TL-gRNA) and the mutating payload plasmid containing the desired mutation, followed by fluorescence-activated cell sorting (FACS) to collect cells that do not express GFP because UKiS marker sequences at both target alleles were replaced by the payload sequence. b Schematic illustration of the two UKiS donors. The UKiS donors consist of homology arms and insulators on both sides and, between them, a marker gene encoding a chimeric protein consisting of GFP and one of two antibiotic markers (puromycin or blasticidin), each of which is linked by a T2A self-cleaving peptide sequence. The sequence corresponding to TL-gRNA partially overlaps with the right arm of the donor sequence.

Fig. 2
Fig. 2. The first step of UKiS: replacement of the first intron of TP53 with the UKiS donor.

a Graphical representation of the human TP53 locus from the UCSC genome browser, indicating one TP53 mRNA variant (NM_000546) and the histone activation mark (H3K27Ac and H3K4Me3), and transcription factor binding regions, all of which were identified by the ENCODE project, and retrotransposon-derived elements (LINEs and SINEs are long and short interspersed nuclear elements, respectively). The regions targeted by the homologous arms of the UKiS donors are denoted by black boxes. The same arms were also used for the mutating payloads during the second step of UKiS. The gRNA-TP53(R) target sequence is highlighted in red and the PAM sequence part is underlined within the sequence of the right arm part. b Schematic diagram of the first step of UKiS for the first intron of TP53. Both UKiS donor plasmids, the marker parts of which are indicated by the same colors as shown in Fig. 1b, were transfected into HCT116 cells, leading to isolation of cell clones that had undergone homologous recombination within the TP53 locus after dual selection with puromycin and blasticidin. Horizontal lines flanked by two arrowheads represent the target regions for the junction genotyping PCR. c Representative gel image of the junction genotyping PCR to confirm deletion of the TP53 first intron and insertion of the UKiS marker, with the expected length of PCR genotyping amplicons indicated. Of the 15 selected clones, 8 had successful replacement of the intron with the UKiS donor in both alleles and are highlighted in red. Source data are provided as a Source Data file.

Fig. 3
Fig. 3. The second step of UKiS: replacement of UKiS donor alleles in the first intron of TP53 with either the wild-type first intron or one of three mutant first introns.

a Schematic diagram of UKiS donor alleles in clone #10 (#1–10), the marker parts of which are indicated by the same colors as shown in Fig. 1b. Horizontal lines flanked by two arrowheads represent the target regions for the junction genotyping PCR performed below. Flow cytometric analysis of GFP fluorescence in clone #1–10. Replacement of both UKiS donor alleles with synthetic introns (b: human TP53 full-length intron, c the entirely deleted intron, (d) syntenic mouse intron, (e) syntenic zebrafish intron) in clone #1–10. First, the mutating payload plasmid and TL-gRNA/Cas9 expression plasmid were transfected into clone #1–10. Thereafter, GFP-negative cells were collected by FACS and cloned. Biallelic substitution of UKiS markers with the mutating payload plasmid was confirmed by junction genotyping PCR that targeted the regions represented by horizontal lines flanked by two arrowheads in the schematic diagrams of the TP53 locus after successful replacement, with the expected length of PCR genotyping amplicons indicated. In the agarose gel images, lane numbers of clones that underwent successful recombination in both alleles are in red. Source data are provided as a Source Data file.

Fig. 4
Fig. 4. Validation of biallelic replacement of the first intron of TP53 in the first and second step of UKiS.

a Graphical representation of the human TP53 locus, indicating the positions and allele frequencies of eight common SNPs (filled orange circles; from dbSNP151). Among them, direct sequencing of PCR amplicons from genomic DNA of the parental HCT116 cells demonstrated that the SNP site rs12947788 is heterozygous in HCT116 (black arrow). Black boxes represent homology arms used in our UKiS mutagenesis to TP53, and the horizontal line flanked by two arrowheads represents the target region for PCR and subsequent sequencing. b Genotyping of clone #1–10, which was used to create all the TP53 mutant clones in this study. Allele-specific PCR was performed by using primers for puromycin or blasticidin marker sequences. The puromycin and blasticidin alleles had C and T at rs12947788, respectively. Horizontal lines flanked by two arrowheads represent the target region for the PCR of each allele. The heterozygous SNP site, rs12947788, is indicated within the 7th intron with filled orange circles. c For human wild-type intron clones #2–1, #2–2, and #2–3, graphical representation of the human TP53 locus is shown on the top: black boxes represent the positions of homology arms used in our UKiS mutagenesis to TP53, the horizontal line flanked by two arrowheads represents the target region for PCR, and the filled orange circles denote the heterozygous SNP site rs12947788. Direct sequencing of the PCR genotyping amplicons indicated double peaks only at rs12947788 on the resultant sequencing chromatograms for these three clones.

Fig. 5
Fig. 5. Evaluating the influence of intron deletion and syntenic substitution of the first intron of TP53 on its expression.

a Representative image of agarose gel electrophoresis for RT-PCR products targeting the TP53 mRNA from three clones of the four variants of the first intron of TP53: human wild-type intron, intron deletion, mouse intron, and zebrafish intron (clone numbering in this figure is as in Figs. 3 and 4). b Schematic drawing of splice site selection of the TP53 first intron in the cell clones with the wild-type human intron (top) and the zebrafish intron (bottom). In the HCT116 cells, the zebrafish intron leads to use of the same 5′ splice site as the human wild-type intron does in most cases, but an AGGT site within the first exon is also used as a minor 5′ splice site. c Real-time RT-PCR of TP53 mRNA. 18S rRNA was used as an internal control. Reactions were run in duplicate in three independent experiments. Clones with the entirely deleted intron and syntenic mouse intron showed increased TP53 mRNA expression as compared with those with the human wild-type intron. The relative fold-changes were calculated by the ΔΔCt method. d Representative image of immunoblotting for TP53 translational expression in the clones shown in (a). Blots of TP53 and α-Tubulin (internal control) were derived from samples run on parallel gels. The blot images for TP53 and α-Tubulin were cropped from each of the membrane (some regions were removed for clarity). Blotting was performed in three independent experiments. e Quantification of TP53 translational expression from the immunoblotting experiments shown in (d). Bands were quantified by ImageJ. Normalization was performed using α-Tubulin expression. Data in (c) and (e) represent the mean ± SD of three independent experiments for the individual clones, and the p-values (in parentheses; relative to the human intron clones) were calculated by a two-tailed Student’s t-test. Reproducibility of (a, c, d, and e) was confirmed in three independent experiments. Source data in (a, c, d, and e) are provided as a Source Data file.

Fig. 6
Fig. 6. Removal of retroelement and Alu sequences from the endogenous first intron of TP53.

a Graphic representation of the synthesis of retroelement- and Alu-free versions of the TP53 first intron. Non-retroelement and non-Alu sequences are shown in red and orange, respectively. b Transcriptional and translational impacts of retroelement and Alu removal on TP53 expression. c Transcriptional impacts of partial removal of Alu elements and removal of non-Alu sequences on TP53 transcription. The removed regions are denoted by dashed lines in the schematic drawings of the 5 deletion mutants. In (b) and (c), three clones of each mutant cell were subjected to real-time RT-PCR (b, c) and immunoblotting (b). 18S rRNA was used as an internal control for real-time RT-PCR. Relative fold-changes were calculated by the ΔΔCt method. For immunoblotting, α-Tubulin was used as an internal control. Data in (b, c) represent the mean ± SD of three independent RT-PCR experiments, and the p-values (in parentheses; relative to the human intron clones) were determined by a two-tailed Student’s t-test, based on three independent experiments for the individual clones. Source data in (b, c) are provided as a Source Data file.

Fig. 7
Fig. 7. UKiS application for iPS cells and longer noncoding targets.

a UKiS for deletion of the first intron of TP53 in iPS cells. In addition to gRNA-TP53(R), the gRNA-TP53(L) was used, target sequence of which is highlighted in red, and the PAM sequence part is underlined. Intron deletion in the second step of UKiS was confirmed by junction genotyping PCR targeting the 3.3 kb region represented by a horizontal line flanked by two arrowheads. Clones having undergone successful intron removal are shown in red. The iPS cell clone used for the second step of UKiS (#1–2) was obtained by the first step of UKiS as shown in Supplementary Fig. 11. b Immunocytochemical staining for expression of pluripotent markers OCT4 (green) and NANOG (red) in the parental iPS cells and in the three mutant clones in which the first intron of TP53 was deleted. Nuclear localization was confirmed by staining with DAPI (blue). Scale bar, 50 μm. Deletion of all introns from three human genes (c: CD44, d: MET, e: APP) using UKiS in HCT116 cells. Deletion of all introns was confirmed by junction genotyping PCR targeting the cDNA sequence from the first exon to the last exon of each gene, as represented by horizontal lines flanked by arrowheads, with the expected length of PCR genotyping amplicons indicated. Clones having successful recombination are shown in red. Representative image of the agarose gel electrophoresis for RT-PCR products targeting the (f) CD44, (g) MET, and (h) APP mRNA from original HCT116 cells, cell clones isolated after the first step of UKiS (Supplementary Figs. 13–15), and three clones corresponding to cells with intron-free genes of interest. The relative fold-changes of gene expression were calculated by quantifying band intensities using ImageJ software. Values of mean ± SD of three independent RT-PCR experiments are shown. Source data are provided as a Source Data file.

Similar articles

Cited by

References

    1. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921. doi: 10.1038/35057062. - DOI - PubMed
    1. Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse genome. Nature. 2002;420:520–562. doi: 10.1038/nature01262. - DOI - PubMed
    1. The ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489:57–74. doi: 10.1038/nature11247. - DOI - PMC - PubMed
    1. Wright JB, Sanjana NE. CRISPR screens to discover functional noncoding elements. Trends Genet. 2016;32:526–529. doi: 10.1016/j.tig.2016.06.004. - DOI - PMC - PubMed
    1. Shukla A, Huangfu D. Decoding the noncoding genome via large-scale CRISPR screens. Curr. Opin. Genet. Dev. 2018;52:70–76. doi: 10.1016/j.gde.2018.06.001. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances