Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System

Abstract

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.

Homologous recombination strategy for introduction of the polyQ expansion in HTT exon 1 into wild type cells.

(a) Homologous donor containing a neomycin resistance cassette 1.5 kb upstream of exon 1 as described previously was used as a donor. Cas9 guide RNA (gRNA) sites were designed near the translational start site of the gene. HTT gRNA sequences were designed based upon criteria described previously. (b) Experimental design for treatment of 293F cells with Cas9 constructs. Cells were lipofected at day 0, placed on selection at day 1 and remained on selection until the cells were harvested at day 18. (c) At day 18 cells were fixed and stained with methylene blue/methanol and scanned on a flatbed scanner. Resulting scans were analyzed with the colony counting function in ImageJ. (d) There was a significant increase in colonies in all cells treated with Cas9, HTT gRNA and donor. This increase was most pronounced in cells expressing WT Cas9 and HTT specific gRNAs. The increase in colony number was donor-dependent.

Pooled 293F neomycin-resistant cells were analyzed by western blot.

(a) To identify 1C2-reactive full-length HTT protein indicative of homologous recombination a western blot assay was utilized. Total HTT (MAB2166) and β-actin are also shown. (b) Quantitated levels of 1C2 signal normalized to β-actin are significantly increased in cells treated with WT Cas9 and HTT specific gRNAs realtive to cells treated with a gRNA targeting a different (AAVS1) locus in the genome. (c) Cas9 D10A show significant increase in HTT gRNA2-mediated recombination relative to control, AAVS1 gRNA assisted cells. (d) 1C2 western blot analysis of individual clones (1-12) transfected with Cas9 D10A, HTT gRNA1, and donor compared with control gRNA and untransfected controls.

Cas9-mediated homologous recombination in iPSCs.

(a) Experimental design and screening process for transfected iPSCs. (b) Candidates for potential targeted integration of HTT exon 1 97Q donor knockin events are screened first by PCR. Separate primer sets specifically amplify either the endogenous CAG length (first lane of each clone) or the introduction of the modified 97Q exon 1 (second lane). Candidates were identified by the loss of one endogenous band coupled with gain of 97Q band. (c) Genomic DNA from iPSC clones are digested with HindIII and probed with a 300 bp radiolabeled probe that is specific to a region external to the targeting donor sequence. Successful integration of targeting donor sequence results in the introduction of a HindIII site that results in truncation of the non-targeted fragment length from 10 kb to 5 kb. (d) Table summarizing frequency of HR and conditions.

Supplemental

(a) Specificity and mismatch scores for HTT gRNA1 and 2 assessed by the CRISPR resources design tool¹⁰. (b) Methylene blue stained colonies of HTT TALEN pair 1 and 2 + donor transfected 293F cells compared to donor alone. (c) Quantification of colony number from HTT TALEN + donor transfected cells compared to donor alone and Cas9/HTT gRNA1 + donor transfected cells.

Supplemental

Pooled 293T resistant cells were analyzed by western blot 6 days after transfection with indicated conditions to identify 1C2-reactive full-length HTT protein indicative of homologous recombination. (a) Western blot confirms 1C2 signal is specific to Cas9 and HTT gRNAs, with little to no signal apparent in untreated, donor alone or donor/Cas9/AAV controls (b) Quantification of western data.