Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9 Journal Article
| Authors: | Paquet, D.; Kwart, D.; Chen, A.; Sproul, A.; Jacob, S.; Teo, S.; Olsen, K. M.; Gregg, A.; Noggle, S.; Tessier-Lavigne, M. |
| Article Title: | Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9 |
| Abstract: | 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 cells. 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). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, 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 (APP Swe) and presenilin 1 (PSEN1 M146V) 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. © 2016 Macmillan Publishers Limited. All rights reserved. |
| Keywords: | adolescent; genetics; mutation; mouse; animal; metabolism; animals; mice; allele; dna repair; alleles; heterozygote; bacteria (microorganisms); genetic engineering; nucleotide sequence; substrate specificity; homozygote; dna breaks, double-stranded; double stranded dna break; base sequence; sequence homology; enzyme specificity; onset age; age of onset; mutagenesis; alzheimer disease; dna cleavage; induced pluripotent stem cells; amyloid precursor protein; presenilin; presenilins; dna template; amyloid beta-protein precursor; templates, genetic; procedures; genetic association studies; genes, dominant; guide rna; rna, guide; induced pluripotent stem cell; humans; human; male; female; secretion (process); crispr cas system; dominant gene; genetic association study; crispr-cas systems |
| Journal Title: | Nature |
| Volume: | 533 |
| Issue: | 7601 |
| ISSN: | 0028-0836 |
| Publisher: | Nature Publishing Group |
| Date Published: | 2016-05-05 |
| Start Page: | 125 |
| End Page: | 129 |
| Language: | English |
| DOI: | 10.1038/nature17664 |
| PUBMED: | 27120160 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 July 2016 -- Source: Scopus |
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