Efficient Introduction of Specific Homozygous and Heterozygous
LETTER doi:10.1038/nature17664 Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9 Dominik Paquet1*, Dylan Kwart1*, Antonia Chen1, Andrew Sproul2†, Samson Jacob2, Shaun Teo1, Kimberly Moore Olsen1, Andrew Gregg1,3, Scott Noggle2 & Marc Tessier-Lavigne1 The bacterial CRISPR/Cas9 system allows sequence-specific gene prokaryotes12. As the efficacy of potential blocking mutations has not editing in many organisms and holds promise as a tool to generate been systematically studied in eukaryotic cells, we tested their effect models of human diseases, for example, in human pluripotent on HDR accuracy in wild-type human induced pluripotent stem stem cells1,2. CRISPR/Cas9 introduces targeted double-stranded cells (iPS cells) (Extended Data Fig. 1) and, for comparison, human breaks (DSBs) with high efficiency, which are typically repaired embryonic kidney (HEK293) cells. We introduced Cas9–eGFP and by non-homologous end-joining (NHEJ) resulting in nonspecific single guide RNA (sgRNA) plasmids together with five pooled repair insertions, deletions or other mutations (indels)2. DSBs may ssODN templates, which in addition to the APPSwe or PSEN1M146V also be repaired by homology-directed repair (HDR)1,2 using pathogenic mutation also contained a putative silent CRISPR/ a DNA repair template, such as an introduced single-stranded Cas-blocking mutation in the PAM or guide RNA target sequence, oligo DNA nucleotide (ssODN), allowing knock-in of specific or a control non-blocking mutation outside those regions (Fig. 1b, c mutations3. Although CRISPR/Cas9 is used extensively to and Supplementary Tables 1 and 2). engineer gene knockouts through NHEJ, editing by HDR remains We analysed genomic loci of Cas9–eGFP-expressing cells by inefficient3–8 and can be corrupted by additional indels9, preventing next-generation sequencing and determined the fraction of HDR its widespread use for modelling genetic disorders through reads that were ‘accurate’, that is, without undesirable indel modi- introducing disease-associated mutations.
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