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.

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

“…The edited iPSC-derived cortical neurons displayed the disease-specific biochemical phenotypes of amyloid-β (A-β) peptide generation [90]. Thus, Alzheimer's disease-associated phenotypes in neurons can be faithfully modeled in vitro using genome editing technology in human iPSCs.…”

“…Using this approach, early-onset Alzheimer's diseasecausative mutations in amyloid precursor protein (APP Swe ) and presenilin1 (PSEN1 M146V ) were precisely introduced into HEK293 cells and iPSCs [90]. The edited iPSC-derived cortical neurons displayed the disease-specific biochemical phenotypes of amyloid-β (A-β) peptide generation [90].…”

“…In the second step, only the secondary mutations are removed by a redesigned guide RNA, which targets the 20-bp sequence containing the introduced CRISPR/Cas9-blocking mutations and the modified repair ssODN template. Alternatively, a Cas9 variant such as VRER-Cas9, which recognizes the modified PAM sequence introduced as a blocking mutation in the first step, can also be used in the second step [90].…”

“…1g) [90,91]. In the first step of this approach, to prevent recutting by CRISPR/Cas9 and unwanted indels subsequently being introduced into human cultured cells using an ssODN template carrying the intended mutation and secondary silent mutations.…”

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

“…A chemical modification of the single-stranded template oligonucleotide, introduced to increase its stability, also favors in ovo HDR (Renaud et al 2016). In addition, a recent study showed that HDR accuracy can be dramatically increased by incorporating silent mutations, along with the desired mutation, to prevent subsequent NHEJ events (Paquet et al 2016). The knowledge of the molecular mechanisms that underlie each DNA repair pathway offers some other interesting possibilities for manipulating the outcome of the repair.…”