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, 10 (1), 4856

Genome-wide Microhomologies Enable Precise Template-Free Editing of Biologically Relevant Deletion Mutations

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Genome-wide Microhomologies Enable Precise Template-Free Editing of Biologically Relevant Deletion Mutations

Janin Grajcarek et al. Nat Commun.

Abstract

The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of human deletion alleles for flanking microhomologies (µHs). a Overview of double-strand break (DSB) DNA repair pathways and their outcomes. homology-directed repair (HDR), classical non-homologous end joining (c-NHEJ), microhomology-mediated end joining (MMEJ). b Schematic of the MHcut tool used to identify the microhomologous sequences. microhomologies (µHs, green) or nested µHs (red), SpCas9 PAM (underline) and DSB location (pink bolt). c Numbers for variant data input and result of filters applied to the MHcut tool output. bp, base pairs. d Distribution of µH-flanked deletion variants by deletion size. e Distribution of µH-flanked deletion variants by µH length. f µH-flanked deletion variants plotted by µH distance with µH length indicated by fill color. g Percentage of protein-coding genes with µH-flanked deletion variant in exonic coding sequences
Fig. 2
Fig. 2
Selected pathogenic target µH-flanked deletion mutations can be recreated with high precision in hiPSCs and hESCs. a Filtered MHcut tool output of potential target pathogenic variants for the parameters shown. Graph at the right shows the distribution of target variants by µH distance with µH length indicated by fill color. b Selected target variant list. µH (green), DSB location (pink bolt), SpCas9 PAM (underline). c Schematic of the experimental method used to create MMEJ deletion alleles in 1383D6 hiPSCs and H1 hESCs. d Overall ratio of indel mutations found in the transfected hiPSC or hESC cell populations. e Ratio of the target MMEJ outcome among total indels. Means ± s.e.m. for n = 3 biological replicates. Source data are in the Source Data file
Fig. 3
Fig. 3
@Target variant associated with muscular dystrophy created by MMEJ recapitulates DYSFERLIN loss-of-function. a Sequence verification of a precise 5 bp deletion mutation in DYSF. Deletion (dotted line), DSB location (pink bolt), µH (green), SpCas9 PAM (underline). b Altered protein sequence of DYSFERLIN caused by the 5 bp deletion mutation. c Schematic of the experimental procedure to induce myogenic differentiation in hiPSCs by overexpression of MYOD from a piggyBac transposon. Differentiation day (D). d Immunostaining for DYSFERLIN and MHC in differentiated hiPSC populations. Comparison of the isogenic parental cell line, three derived clones carrying the disease mutation and a muscular dystrophy patient derived hiPSC cell line. Scale bar indicates 100 µm; ratio of mCherry + cells measured by FACS in corresponding electroporated (EP) hiPSC populations indicated on the right
Fig. 4
Fig. 4
Erythropoietic protoporphyria disease models created by MMEJ display ALAS2 gain-of-function and FECH loss-of-function. a Phenotypic consequences of ALAS2 gain-of-function and FECH loss-of-function mutations on the accumulation of metabolites in the heme synthesis pathway. 5-Aminolevulinic acid (5-ALA), Protoporphyrin IX (PPIX). b Altered protein sequences of ALAS2 and FECH caused by deletion mutations. c Sequence verification of a precise 4 bp deletion mutation in ALAS2 and a 5 bp deletion in FECH generated by either plasmid or RNP transfection. Deletion (dotted line), DSB location (pink bolt), µH (green), SpCas9 PAM (underline). d Schematic for cell culture conditions during erythroid differentiation. Differentiation day (D). e Ratio of erythroid cells in differentiated hiPSC populations on D26, as indicated by CD235a-FITC and CD71-APC markers, measured by FACS. f Gene expression levels measured by qRT-PCR of ALAS2 and FECH in undifferentiated cells and D26 cell populations normalized to cord blood cells. g PPIX-Qdot® 605 fluorescence intensity in erythroid cell populations of mutant and normal cell lines on day 26, measured by FACS. h Dose-response curve of PPIX accumulation in response to 5-ALA treatment in undifferentiated hiPSC disease model clones and the isogenic parental line. Median fluorescence intensity of PPIX-Qdot® 605 measured by FACS and normalized to the parental cell line at 0.5 mM 5-ALA. Means ± s.d. for n = 3 biological replicates. Source data are in the Source Data file
Fig. 5
Fig. 5
MHcut tool complements existing tools for selecting a suitable gene editing target and gRNA. (*MHcut tool contains the editing prediction for the target deletion from the inDelphi editing outcome prediction tool)

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