Viable Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches

Abstract

Here, we describe an expansion of the typical DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus by zygotic injection. Driving our efforts was the desire to create a whole animal model that would replace 17 kilobase pairs (kbp) of the mouse Bcl2l11 gene with the corresponding 25-kbp segment of human BCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3' of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic embryonic stem cell (ESC) targeting, dual selection, and recombinase-driven cassette removal (ESC/Blastocyst Approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with redundant single guide RNAs (sgRNAs) and Cas9 mRNA into mouse zygotes (CRISPR/Zygote Approach). In both instances, we were able to achieve humanization of Bcl2l11 to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, 2.9-kbp intronic segment.

Figure 1

Structure of Human and Mouse BCL2L11/Bcl2l11 Genes. A simplified schematic of the two gene structures [Mouse Bcl2l11, University of California-Santa Cruz (UCSC) transcript isoform 1; human BCL2L11, UCSC transcript isoform 1] is shown. Driving the design of this engineering experiment was the desire to humanize the central region of the mouse Bcl2l11 gene to investigate a cancer-associated 2.9-kbp deletion polymorphism.

Figure 2

(a) ESC/Blastocyst Approach in Mouse Embryonic Stem Cells. The mouse Bcl2l11 locus, a gene targeting vector (pTLD39), and the modified locus are shown. A gene-targeting vector/donor molecular was constructed placing a 25-kbp segment of the human BCL2L11 gene between mouse homology arms, placing removable selectable marker cassettes at each end of the human segment, and placing loxP sites around a 2.9-kbp segment of human DNA deleted in 12% of the East Asian population (vector names, blue pTLD labels; guide binding areas, orange stars; genotyping oligonucleotide binding sites, oTLD-labelled arrows; proximal junction on mouse locus, mPJ; distal junction on mouse locus, mDJ; proximal mouse/human junction, PJ; distal mouse/human junction, DJ; Targeted Locus Amplification capture amplimers; circled numbers). See text for details. (b) CRISPR/Zygote Approach in Mouse Zygotes. The mouse Bcl2l11 locus, a gene targeting vector (pTLD67), and the modified locus are shown. Vector is as in “a.” above after removal of the Neo^R and Puro^R selection cassettes that are not necessary with the CRISPR/Zygote Approach (vector names, blue pTLD labels; genotyping oligonucleotide binding sites, oTLD-labelled arrows; proximal junction on mouse locus, mPJ; distal junction on mouse locus, mDJ; proximal mouse/human junction, PJ; distal mouse/human junction, DJ; junction over the deletion, ΔJ). See text for details.

Figure 3

Genotyping. Genotyping Assays for various genotypes of mice from our CRISPR/Zygote Approach are shown. (a) Multiplex genotyping with the mPJ and PJ assays identifies the proximal mouse/mouse and mouse/human junctions, respectively. (b) Multiplex genotyping with the mDJ and DJ assays identifies the distal mouse/mouse and mouse/human junctions, respectively. (c) Genotyping with the ΔJ assay identifies a 17-kbp deletion within the mouse Bcl2l11 gene. (d) Genotyping with the P2.9 assay identifies the 5′ end of the 2.9-kbp polymorphic segment. (e) Genotyping with the D2.9 assay identifies the 3′ end of the 2.9-kbp polymorphic segment. (f) Genotyping with the Δ2.9 assay identifies Cre-loxP-mediated deletion of the 2.9-kbp polymorphic segment. BCL2L11^h, humanized BCL2L11 allele; Bcl2l11^m, wildtype mouse Bcl2l11 allele; Bcl2l11Δ, mouse Bcl2l11 deletion allele; BCL2L11Δ^2.9, humanized BCL2L11 2.9-kbp deletion allele. Gel images are cropped.

Figure 4

Sequencing Small Insertions/Deletions (INDELs) Among CRISPR/Zygote Approach Founder (P₀) Mice. Sanger sequencing chromatograms and deduced sequences are shown for five potential founder (P₀) mice (P₀ #s 4, 6, 9, 17, and 19) from the CRISPR/Zygote Approach. Red arrows, location of INDELs within chromatograms; red bracket, location of double peaks within chromatograms; red rectangles, location of INDELs within deduced sequence; red dots, location of double peaks within deduced sequence; vertical red lines, point of DSB creation by Guides G7 and G8; green PAM, location of NGG PAM sites; black dots, deviations from wildtype sequence which is also shown.

Figure 5

Digital Droplet PCR (ddPCR) Assessment of Homology Arm and Bcl2l11/BCL2L11 Segment Copy Number. Two dimensional amplitude plots for assessing copy number of the 5′ homology arm, mouse Bcl2l11 gene, human BCL2L11 gene, and 3′ homology arm by ddPCR are shown. For any analyzed DNA with a genotype of interest, the fluorescence amplitude within droplets, and the relative number of droplets can be used to determine the relative copy number of two DNA segments, in this case, elements of the BCL2L11 humanization and a β-actin control. See Table 6 and text for more detail.

Figure 6

Linkage analysis of the BCL2L11 integration site following CRISPR-stimulated homologous recombination in mouse zygotes. Shown is the linkage analysis for 22 F₂ progeny of a C57BL/6NJ X FVBB6NF1/J-BCL2L11 backcross and 28 F₂ progeny of an FVB/NJ X FVBB6NF1/J-BCL2L11 backcross. Linkage and haplotype analyses indicate that the BCL2L11 vector’s integration has occurred between markers rs4223406 and rs3689600 and its segregation is fully concordant with markers rs13476756 and rs3662211. This result is entirely consistent with integration of the human BCL2L11 segment within the endogenous mouse Bcl2l11 gene as designed. Megabasepair positions along Mouse Chromosome 2, the position of mapping SNPs, and genetic distances are shown. Concordant markers are enclosed in a rectangle. The extent of the Bcl2l11/BCL2L11 critical interval (within which the humanization vector integration is mapped) is shown in green.

Figure 7

(a) Targeted Locus Amplification — Chromosomal Amplification. High-throughput sequencing reads, obtained through the use of the TLA technique with human and mouse BCL2L11-/Bcl2l11-derived target amplimers, identify distal Chr2 as the integration site of the humanizing gene targeting vector/donor molecule. This result is entirely consistent with integration of the human BCL2L11 segment within the endogenous mouse Bcl2l11 gene as designed (vertical axis, read-depth along each of the mouse chromosomes; horizontal axis, chromosomal position). (b) Targeted Locus Amplification — Reads Mapped to the Mouse Genome. 1. High-throughput sequencing reads, obtained through the use of the TLA technique with human and mouse BCL2L11-/Bcl2l11-derived target amplimers, localize to the mouse Bcl2l11 locus outside of the humanized region. This result is entirely consistent with integration of the human BCL2L11 segment within the endogenous mouse Bcl2l11 gene as designed (vertical axis, read-depth along the mouse Bcl2l11 locus for each of three target amplimers; horizontal axis, chromosomal position; red lines, regions of the two mouse homology arms; red arrows, internal boundary of homology arms and location of chimeric human/mouse BCL2L11/Bcl2l11 sequencing reads; central blue gene structure, Bcl2l11; left blue gene structure, an adjacent gene; narrow vertical rectangle, region examined in panel 2). 2. Sequencing reads mapping to the 5′ end of the 5′ homology arm/flanking mouse genome boundary. Sequencing reads spanning the homology arm/genome boundary are contiguous (horizontal pink and purple bands). Fusion reads (horizontal bands with multicolored segments) arise only from nearby NlaIII sites (NlaIII-labelled vertical blue lines) and are an artifact of the TLA technology. No fusion reads suggest integration at an ectopic locus. Analysis of the 3′ end of the 3′ homology arm was similar (not shown). (c) Targeted Locus Amplification — Chimeric Reads. Representative reads from the 5′ and 3′ mouse/human breakpoints (junctions) of the humanized BCL2L11/Bcl2l11 locus (red lettering, mouse-derived sequence; black lettering, human-derived sequence; blue lettering, vector-derived HindIII site at the 5′ mouse/human junction; green lettering, vector-derived EcoRI, attL, and BamHI sites at the 3′ mouse/human junction; underlined purple lettering, additional vector-derived sequences). (d) Targeted Locus Amplification — Reads Mapped to the Vector/Donor Sequence. 1. High-throughput sequencing reads, obtained through the use of the TLA technique with human and mouse BCL2L11-/Bcl2l11-derived target amplimers, localize across the pTLD67 gene targeting vector/donor molecule including mouse and human elements. This result is entirely consistent with integration of the human BCL2L11 segment within the endogenous mouse Bcl2l11 gene as designed (vertical axis, read-depth along the pTLD67 vector for each of three target amplimers; horizontal axis, position within the vector; red lines, regions of the two mouse homology arms; narrow vertical rectangle, region examined in panel 2). 2. Sequencing reads mapping to the 5′ end of the 5′ homology arm/vector boundary. Reads arising from the point of integration (horizontal bands with multicolored segments) appear as fusion reads at the homology arm/vector boundary. Additional fusion reads arise only from a nearby NlaIII site (NlaIII-labelled vertical blue line) and are an artifact of the TLA technology. No fusion reads suggest continuity into the vector’s backbone. Analysis of the 3′ end of the 3′ homology arm was similar (not shown).