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Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9


The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells1,2. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels)2. DSBs may also be repaired by homology-directed repair (HDR)1,2 using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations3. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient3,4,5,6,7,8 and can be corrupted by additional indels9, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed ‘CORRECT’ for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation’s incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer’s disease-causing mutations in amyloid precursor protein (APPSwe)10 and presenilin 1 (PSEN1M146V)11 and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

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Figure 1: CRISPR/Cas-blocking mutations increase HDR accuracy by preventing re-editing and can be used for scarless CORRECT editing.
Figure 2: A monotonic inverse relationship between mutation incorporation and distance from the CRISPR/Cas9 cleavage site.
Figure 3: Introduction of heterozygous or homozygous mutations into iPS cells by manipulating the cut-to-mutation distance or using mixed HDR templates.
Figure 4: APPSwe and PSEN1M146V knock-in lines display genotype-dependent disease-associated changes in Aβ secretion.


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This research was supported by The Rockefeller University, The New York Stem Cell Foundation, The Ellison Foundation, Cure Alzheimer’s Fund, the Empire State Stem Cell fund through new York State Department of Health contract number C023046, and CTSA, RUCCTS grant number 8 UL1 TR000043 from the National Center for Advancing Translational Sciences (NCATS, NIH). D.P. is a New York Stem Cell Foundation Druckenmiller Fellow and received a fellowship from the German Academy of Sciences Leopoldina. D.K. is a Howard Hughes Medical Institute International Student Research Fellow and received a fellowship from the National Sciences and Engineering Research Council of Canada. S.T. is supported by the Agency for Science, Technology and Research of Singapore. A.G. is supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-institutional MD-PhD program. We thank members of the Tessier-Lavigne laboratory and L. Marraffini for discussions. Our thanks also go to S. Mazel and the team at the Rockefeller Univeristy Flow Cytometry Resource Center, J. Gonzalez and the team at the Rockefeller University Translational Technology Core Laboratory, C. Zhao and the team at the Rockefeller University Genomics Resource Center, and D. Paull and M. Duffield for technical help. Opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Fund, the New York State Department of Health, or the State of New York. The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations



D.P., D.K. and M.T.-L. conceived and designed the study. D.P. and D.K. performed and analysed the experiments. A.C. and A.G. helped perform the experiments. S.T. helped analyse next-generation sequencing data. A.S., S.J. and S.N. generated and characterized the iPS cells. K.M.O. performed and analysed the electrophysiology assays. D.P., D.K., and M.T.-L. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Marc Tessier-Lavigne.

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Competing interests

A patent application relating to this work has been filed by D.P., D.K. and M.T.-L.

Extended data figures and tables

Extended Data Figure 1 In vitro and in vivo characterization of the wild-type 7889SA human iPS cell line.

a, Immunofluorescence staining of pluripotent stem cell markers. b, iPS cells possess a normal human male karyotype. c, Nanostring expression analysis of pluripotent stem cell genes in reprogrammed iPS cells compared to HUES9. d, In vivo differentiation and analysis of iPS-cell-derived teratoma containing tissues of all germ cell layers. Scale bars, 100 μm.

Extended Data Figure 2 CRISPR/Cas-blocking mutations increase HDR accuracy by preventing re-editing, are incorporated in multiple rounds of re-editing and can also be applied to scarless editing using CORRECT.

a, b, HDR reads from five unpooled templates containing intended pathogenic and CRISPR/Cas-blocking or non-blocking control mutations. Percentages of accurate HDR for reads containing blocking (B) or control (C) mutations at the APP (a) and PSEN1 (b) locus in HEK293 cells. Values represent mean ± s.e.m. (n = 3). ND, not detected. ***P < 0.001, **P < 0.01, one-way ANOVA. c, d, Proportion of next-generation sequencing reads containing putative single, double, or triple HDR events (left) for APP (c) and PSEN1 (d). Putative ‘double HDR’ examples of the most frequent reads that either contain a non-blocking control mutation C with an additional CRISPR/Cas-blocking mutation B, or do not contain C and have two different CRISPR/Cas-blocking mutations (middle). Reads that contain the non-blocking mutation (C+) are more frequently re-edited to incorporate a CRISPR/Cas-blocking mutation (‘double HDR’) than reads containing a blocking mutation B instead of the non-blocking mutation C (C−). See Fig. 1c for legend. To facilitate data analysis, all replicates were pooled to increase read numbers for rare events. e, f, Schematics depicting details of the two tested CORRECT approaches: in step 1 of re-guide (e), the APPSwe mutation was introduced together with a CRISPR/Cas-blocking guide RNA target mutation, which was then removed again in step 2 using a re-sgRNA specific for the mutated sequence and wild-type Cas9. In step 1 of re-Cas (f), the APPA673T mutation was introduced together with a CRISPR/Cas-blocking PAM-altering NGCG mutation, which was then removed in step 2 using the VRER Cas9 variant, which specifically detects the NGCG PAM. We chose to use the very active APP-sgRNA12 to test CORRECT by re-Cas, which was also used in Fig. 3c and 3d to generate APPSwe mutant lines. However, as the APPSwe mutation is located in the target sequence of this sgRNA, it may block re-editing by CRISPR/Cas and could therefore complicate the interpretation of results. We therefore decided to knock-in the protective APPA673T mutation49 instead, which lies outside of the target sequence. In both cases, the blocking mutations were removed using a CORRECT ssODN repair template, which restored the original sequence at the site of the blocking mutation (which blocks further re-cutting in this step), but retained the intended APP mutation. Note that due to repeated editing, CORRECT may increase the probability of off-target effects, but presumably not the number of potential off-target sites, as the same (for re-Cas) or a very similar (for re-guide) guide RNAs are used in both editing steps.

Extended Data Figure 3 Analysis of CRISPR/Cas9-induced indels in gene edited iPS cells and HEK293 cells.

a, Plot depicting frequency of indels at each position around the targeted locus in all next-generation sequencing reads with editing events from the analysis shown in Fig. 1. Insertions are plotted at the location where they begin, and deletions are plotted across all deleted base positions (top). Histogram illustrating distribution of indel sizes (bottom). b, Indel position (top) and size (bottom) of indel-containing alleles from single-cell clones analysed in Extended Data Fig. 4a, b.

Extended Data Figure 4 Heterozygous clones with HDR on one allele almost always contain indels on the non-HDR allele, and longer ssDNA or dsDNA HDR repair templates do not influence mutation incorporation probabilities related to cut-to-mutation distance.

a, Sanger sequencing reads of both APP alleles of a single-cell clone with mono-allelic HDR (blue arrow). The non-HDR allele is altered by NHEJ in the guide RNA target sequence (orange arrow). b, Single-cell clones with HDR on one allele are mostly altered by NHEJ on the non-HDR allele (APP, n = 26; PSEN1, n = 34). c, Schematic describing the generation of large ssDNA and dsDNA HDR repair templates for the PSEN1 locus (see Methods for details). d, The monotonic relationship between incorporation of intended mutations (M) by HDR and cut-to-mutation distance is not altered by providing longer ssDNA and dsDNA templates (n = 2). Red dashed trend line shows previously determined 100-nt oligonucleotide scan result (from Fig. 2d) for comparison.

Extended Data Figure 5 Mutation incorporation rates at various cut-to-mutation distances follow the distance effect, and mixed repair templates as a strategy to generate heterozygous iPS cell single-cell clones.

a, b, Incorporation rate of APP and PSEN1 pathogenic mutations at increasing distance from the cut site targeted by three distinct sgRNA/ssODN pairs is governed by distance. Incorporation rates (solid dots represent mean ± s.e.m., note s.e.m. is too small to be visible, (n = 3)) match almost exactly the curves for each locus previously determined by oligonucleotide scan (dashed trend line ± s.d. of raw data from Fig. 2c, d). ***P < 0.001, one-way ANOVA. c, d, Mixed ssODN editing approach at the APP locus with blocking mutations in one (c) or both (d) ssODNs (top); zygosity quantification of single-cell clones (d, bottom left) and incorporation rates of CRISPR/Cas-blocking mutation B and pathogenic mutation M determined by next-generation sequencing analysis (d, bottom right). Note that for the M/B approach in c, both oligonucleotides are incorporated at equal levels, as they have similar blocking activities, whereas for the M+B/B approach in d, the M+B ssODN is preferentially incorporated, presumably due to a synergistic blocking effect of both M and B. For the clone quantification in Fig. 3d, the rate of wild-type clones was not assessed, because the silent mutation did not introduce a restriction site. However, given the ~50% ssODN incorporation rates determined by deep sequencing, about 25% of HDR clones are predicted to be wild type. e, Mixed ssODN editing approach at the PSEN1M146V locus (top). Using an sgRNA with the smallest possible cut-to-mutation distance (PSEN1-sgRNA5), two ssODNs were provided, each containing the same silent PAM-altering CRISPR/Cas-blocking mutation B, but only one containing the pathogenic mutation M. Frequencies of pathogenic mutation genotypes in single-cell clones with bi-allelic HDR of B (bottom left) and incorporation rates of CRISPR/Cas-blocking and pathogenic mutations by next-generation sequencing (bottom right). Note that due to the 9 bp distance to the cleavage site, the incorporation of M is lower than 50% (as expected from the distance effect).

Extended Data Figure 6 Characterization of iPS-cell-derived cortical neurons.

a, b, Sanger sequencing reads of APPSwe and PSEN1M146V gene edited iPS cell lines. ce, Immunofluorescence staining of markers for neural precursors at DIV10 (c), cortical neurons at DIV65 (d) and functional synapses at DIV65 (e). Scale bars; 100 μm (c, d), 10 μm (e). f, Evoked action potentials recorded in a neuron current-clamped to −65 mV. g, Mean (±s.e.m.) resting membrane potential (Vrest), action potential threshold and action potential overshoot (DIV 71–85; n = 18). Properties of the largest action potential elicited in each cell were measured. h, Mean number of evoked action potentials increases with increasing stimulus strength. i, Spontaneous synaptic activity recorded in a neuron voltage-clamped to −70 mV. j, Mean (± s.e.m.) frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) (DIV 71–85; n = 8).

Extended Data Figure 7 Possible mechanism underlying the distance effect for HDR-mediated mutation incorporation with CRISPR/Cas9.

CRISPR/Cas9 causes a DSB at a genomic locus, which leads to variable size deletions or strand resections in different cells. Genomes with small deletions or resections are more common than large ones, which is reflected in the distribution of deleted bases after NHEJ (top left). During HDR, only the part of the repair template overlapping this deletion may be used, which results in fewer mutations incorporations more distal to the cleavage site (bottom left, data pooled for APP and PSEN1 from Fig. 2d).

Extended Data Figure 8 Next-generation sequencing data analysis pipeline for HDR and indel detection.

a, For all next-generation sequencing experiments, raw forward and reverse paired next-generation sequencing reads were first merged to obtain single high-quality reads (tool: PEAR), de-multiplexed to separate experiment-specific barcoded reads (seqtk) then filtered to remove low-quality reads. b, For experiments using pooled oligonucleotides containing CRISPR/Cas-blocking mutations (displayed in Fig. 1), reads were separated into wild-type (WT) and edited reads, which were then filtered to include only reads that had incorporated the pathogenic mutation (M+) (that is, containing a pathogenic and CRISPR/Cas-blocking mutation). To account for multiple HDR events after re-editing, reads were then separated into 32 unique categories covering every possible combination of CRISPR/Cas-blocking mutations. c, Reads were aligned (bwa mem) and accurate HDR (perfect alignment) or indel distribution was reported (bam-readcount, R). For analysis in Extended Data Fig. 2c, d, reads that had incorporated multiple CRISPR/Cas-blocking mutation were separately analysed. d, For the mutation incorporation analyses performed in all other figures reads were filtered for the expected sequence and counted.

Extended Data Table 1 List of HDR rates determined by next-generation sequencing and single-cell clone analysis
Extended Data Table 2 Off-target analysis of knock-in APPSwe and PSEN1M146V iPS cell lines

Supplementary information

Supplementary Data

This file contains Supplementary Table 1, a list of APPSwe / PSEN1M146V sgRNAs and primers used for amplification of targeted loci. (XLSX 43 kb)

Supplementary Data

This file contains Supplementary Table 2, a list of ssODNs used as repair templates and enzymes used for RFLP analysis. (XLSX 53 kb)

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Paquet, D., Kwart, D., Chen, A. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

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