A versatile platform for locus-scale genome rewriting and verification

Abstract

Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.

Keywords: genome engineering; genome writing; regulatory genomics; stem cells; synthetic biology.

Fig. 1.

Engineering the HPRT1 locus in hESCs. (A) Replacement of the 42-kb HPRT1 locus in H1 hESCs with an LP (LP-TK) utilizing CRISPR/Cas9 and 1-kb HAs (gray). Cells are selected for LP-TK presence with puromycin and HPRT1 inactivation with 6-TG. (B) PCR genotyping of H1 clones for novel left (L) and right (R) junctions (Jx) using primers illustrated in A. Par, parental H1. (C) Sequencing verification pipeline using WGS or targeted libraries. Capture-seq enriches for regions of interest using biotinylated bait prepared using nick translation from relevant DNA constructs. (D) WGS of parental H1 hESCs and LP-TK clone 58I mapped to hg38 shows the 42-kb deletion of the HPRT1 locus. (E) Mapping to LP-TK (Left) and LP-TK backbone (Right) confirms specific gain of LP-TK; regions cross-mapping with human genome are shaded gray [pEF1α, EEF1A1 promoter; ERT2, ESR1 ligand binding domain (59); pA, EIF1 pA signal]. (F) Mapping to pCas9 confirms plasmid loss; regions shaded gray cross-map with human (pU6, U6 promoter) and LP-TK (PuroR, puromycin-resistance gene). (G) LP-TK at HPRT1 undergoes RMCE with PL1 following transfection and Cre induction. Payload integration can be selected for with blasticidin and GCV. (H) Genotyping of untransfected LP-TK hESCs (clone 58I), PL1-transfected pool, and PL1 clones using PCR primers flanking payload lox sites (illustrated in G). All clones produced the expected 3-kb product (a 5.7-kb product for LP-TK cells was not detected at this extension time). (I) Capture-seq analysis of chosen H1 PL1 clones mapped to PL1 (Left) and its backbone (Right). (J) Capture-seq reads mapped to LP-TK, validating LP loss in PL1 clones. Cross-mapping sequences are shaded gray.

Fig. 2.

Development of an efficient counterselection strategy. (A) Parental (TK⁻) and LP-TK (TK⁺) H1 hESCs were cocultured at the indicated ratios, treated with 1 µM GCV for 4 d, and assayed for the number of live cells using PrestoBlue. Cell counts are shown relative to unmixed parental cells. Bars show mean + SD (n = 2). (B) GCV enters TK⁺ cells and is metabolized into the toxic membrane-impermeable compound GCV-TP, which diffuses into neighboring cells and induces bystander cell death in TK⁻ cells. (C) Big-IN counterselection strategy using PIGA/proaerolysin. (D) Parental and ΔPIGA H1 hESCs cocultured at the indicated ratios for 3 d were treated with 1 nM proaerolysin for 1 d and stained with Crystal violet 3 d later.

Fig. 3.

Allele-specific engineering of the murine Sox2 locus. (A) Replacement of a 143-kb region of the Sox2 locus on the BL6 allele of chromosome 3 (black) in BL6xCAST ΔPiga mESCs with of LP-PIGA utilizing CRISPR/Cas9, facilitated by 0.15-kb HAs (gray). (B, Upper and Lower Left) Screening of BL6xCAST LP-PIGA clones using PCR genotyping primers targeting novel junctions (L Jx and R Jx) illustrated in A. (Lower Right) Secondary screening of 16 clones positive for both junctions using primers for plasmid origin of replication (Ori) and the BL6 Sox2 allele (Sox2[BL6]). Seven clones marked with asterisks had the desired genotype; Clone A1 was selected for further analysis. L, ladder; Par, parental BL6xCAST mESCs. (C–E) Capture-seq analysis of parental ΔPiga BL6xCAST mESCs, LP-PIGA clone A1, and an example failed clone from an independent LP-PIGA delivery. Reads were mapped to the references indicated above. Cross-mapping sequences are shaded gray.

Fig. 4.

Efficient delivery to mESCs. (A) Delivery of three payloads to BL6xCAST ΔPiga LP-PIGA mESCs. (B) PCR genotyping of PL1 (Upper) and Sox2^46kb-MC (Lower) mESC clones for novel junctions illustrated in A. E, empty well; L, ladder. (C and D) Capture-seq analysis of chosen PL1 and Sox2^46kb-MC mESC clones, with Parental and LP-PIGA mESCs as controls. (C) Sequencing coverage mapped to PL1. pEF1α (shaded gray) is present in both LP-PIGA and PL1. (D) Gain of coverage in Sox2^46kb-MC mESCs at the 46-kb payload region. Black ticks under each coverage track indicate detection of BL6 alleles at known SNVs. Internal payload duplication marked in Clone C9 (SI Appendix, Fig. S6). (E) PCR genotyping of Sox2^143kb clones for BL6-specific junctions and loss of LP-PIGA, as illustrated in A. (F) Sox2^143kb mESCs show restored coverage of the full 143-kb genomic region corresponding to the payload. Black ticks under each coverage track indicate detection of BL6 alleles at known SNVs. Coverage at right shows no retention of payload backbone. Cross-mapping sequences are shaded gray. (G) qRT-PCR expression analysis of Sox2^143kb clone G11 and LP-PIGA mESCs for mRNAs from BL6 and CAST Sox2 alleles, payload-derived BSD, and LP-harbored hmPIGA. Bars represent mean + SD for technical replicates (n = 3).

Fig. 5.

Bamintersect, a tool for integration site analysis. (A) Schematic of the bamintersect analysis pipeline. (B–H) Bamintersect results between genomic and custom references indicated at top of each panel. Bars represent the number of reads supporting each junction, normalized to 10 million sequenced reads. Results were annotated as targeted left junction (green), targeted right junction (red), or off-target (blue). For PL1 integration at both HPRT1 and Sox2 (B and D), the targeted left junction is not distinguishable due to its near identity with the LP sequence being replaced. For integrations at Sox2 (C, E, and F), the targeted left junction is adjacent to a low mappability region composed of simple repeats and an Alu sequence, consistently yielding fewer reads relative to the right junction. Allelic analysis in F categorizes reads at expected left and right junctions using known BL6xCAST SNVs; uninformative reads do not overlap known variants.

Fig. 6.

Targeted locus-scale genome rewriting using Big-IN. An allele of interest is replaced by a LP using CRISPR/Cas9-mediated HDR. A pair of gRNAs target the termini of the replaced allele and the LP, and short HAs mediate precise LP integration. Puromycin selects for LP-harboring cells. Next, Cre-mediated recombination of two pairs of heterotypic loxM and loxP sites results in LP/payload cassette exchange and resistance to either GCV for HSV1-ΔTK LPs, or proaerolysin for hmPIGA LPs in cells where endogenous PIGA is inactivated. Positioning the blasticidin cassette (BSD) within the payload permits election for high-efficiency integration; positioning BSD on the payload backbone permits transient selection for scarless delivery. Additionally, backbone HSV-ΔTK (Left) can be counterselected with GCV to limit off-target integration. Each engineering step is comprehensively verified by PCR genotyping, WGS or Capture-seq, and functional assays.