Guidelines for Fluorescent Guided Biallelic HDR Targeting Selection With PiggyBac System Removal for Gene Editing

Abstract

The development of new and easy-to-use nucleases, such as CRISPR/Cas9, made tools for gene editing widely accessible to the scientific community. Cas9-based gene editing protocols are robust for creating knock-out models, but the generation of single nucleotide transitions or transversions remains challenging. This is mainly due to the low frequency of homology directed repair, which leads to the screening of a high number of clones to identify positive events. Moreover, lack of simultaneous biallelic modifications, frequently results in second-allele indels. For example, while one allele might undergo homology directed repair, the second can undergo non-homologous end joining repair. Here we present a step-wise protocol for biallelic gene editing. It uses two donors carrying a combination of fluorescent reporters alongside homology arms directed to the same genomic region for biallelic targeting. These homology arms carry the desired composite of modifications to be introduced (homozygous or heterozygous changes). Plus, the backbone of the plasmid carries a third fluorescent reporter for negative selection (to discard random integration events). Fluorescent selection of non-random biallelic targeted clones can be performed by microscopy guided picking or cell sorting (FACS). The positive selection module (PSM), carrying the fluorescence reporter and an antibiotic resistance, is flanked by inverted terminal repeats (ITR) that are recognized by transposase. Upon purification of the clones correctly modified, transfection of the excision-only transposase allows the removal of the PSM resulting in the integration of only the desired modifications.

Quality Control Strategy for CRISPR-Cas9-Based Gene Editing Complicated by a Pseudogene

“…This mutation was either corrected or inserted at the heterozygous state in patient-derived iPSCs in order to model its implication in PD. The gene editing strategy is based on the protocol published earlier this year by Jarazo and colleagues (Jarazo et al, 2019). The process makes use of two donor vectors for homology directed repair allowing deterministic identification of the gene editing status by a fluorescence-based approach ( Figure 1A).…”

“…From both of these vectors, we amplified the homology arms to be cloned into the final donor vector around the PSM ( Figure 1A and Figure S2). Briefly, the homology arms were amplified from the TOPO vectors described above with overhangs matching the donor vector scaffold after HpaI enzymatic digestion ( Figure S2A, Table S1) (Jarazo et al, 2019). The right homology arm (RHA) was generated in two steps allowing introduction of a TTAA motif and mutation of the PAM sequence.…”

“…The PAM sequence was modified by silent mutation G > C ( Figure S2G). The assembly of the arms into the donor vector scaffold was performed as described ( Figures S2H, I) (Jarazo et al, 2019). At the end of this process, three donor vectors were obtained: one c a r r y i n g t h e m u t a t i o n a s s o c i a t e d w i t h d T o m a t o (dTomato_AGC), one carrying the WT codon associated with dTomato (dTomato_AAC) and one carrying the WT codon associated with EGFP (EGFP_AAC).…”

“…Recently, CRISPR-Cas9 technology emerged as an accessible, reliable, and efficient gene editing method for insertion or correction of point mutations (Arias-Fuenzalida et al, 2017). Therefore we undertook the editing of GBA in patient-derived iPSCs by FACS-assisted CRISPR-Cas9 technology (Jarazo et al, 2019).…”

Parkinson’s disease phenotypes in patient specific brain organoids are improved by HP-β-CD treatment

“…(Arias- Fuenzalida et al, 2017;Jarazo et al, 2019). Briefly, for designing the gRNAs targeting sequence //portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) and inserted into pX330 vector (Addgene 42230) as described in(Ran et al, 2013).Donor constructs were assembled by introducing the correspondent homology arms into donor scaffold containing a positive selection module (PSM) and either EGFP or dTomato (PSM-EGFP and PSM-dTomato) and a blue florescence in the backbone of the plasmid for detecting random integrations (tagBFP), using Gibson assembly.…”