A Universal Surrogate Reporter for Efficient Enrichment of CRISPR/Cas9-Mediated Homology-Directed Repair in Mammalian Cells

Abstract

CRISPR/Cas9-mediated homology-directed repair (HDR) can be leveraged to precisely engineer mammalian genomes. However, the inherently low efficiency of HDR often hampers to identify the desired modified cells. Here, we developed a novel universal surrogate reporter system that efficiently enriches for genetically modified cells arising from CRISPR/Cas9-induced HDR events (namely, the "HDR-USR" system). This episomally based reporter can be self-cleaved and self-repaired via HDR to create a functional puromycin selection cassette without compromising genome integrity. Co-transfection of the HDR-USR system into host cells and transient puromycin selection efficiently achieves enrichment of HDR-modified cells. We tested the system for precision point mutation at 16 loci in different human cell lines and one locus in two rodent cell lines. This system exhibited dramatic improvements in HDR efficiency at a single locus (up to 20.7-fold) and two loci at once (42% editing efficiency compared to zero in the control), as well as greatly improved knockin efficiency (8.9-fold) and biallelic deletion (35.9-fold) at test loci. Further increases were achieved by co-expression of yeast Rad52 and linear single-/double-stranded DNA donors. Taken together, our HDR-USR system provides a simple, robust and efficient surrogate reporter for the enrichment of CRISPR/Cas9-induced HDR-based precision genome editing across various targeting loci in different cell lines.

DNA templates with blocked long 3ʹ end single-stranded overhangs (BL3SSO) promote bona fide Cas9-stimulated homology-directed repair of long transgenes into endogenous gene loci

“…Although the number of GFP-positive cells from ISG-targeting experiments was less than 0.1%, ISGs are expected to be one of the more challenging loci for HDR because IFN-induction has to open up the chromatin for Cas9 and BL3SSO templates to access. Other recent studies ( He et al 2016a , 2016b ; Cai et al 2019 ) measuring low efficiencies of Cas9-stimulated HDR of GFP transgene insertions in human cell lines including HEK293 cells are in accordance with our findings, yet contrast against other studies reporting higher efficiencies of GFP transgene insertions into safe-harbor loci like ROSA26 and AAVS1 ( Ye et al 2018 ; Jayavaradhan et al 2019 ; Wierson et al 2020 ; Yan et al 2020 ), or in animals where embryonic cells are subjected to transgene and Cas9 injection ( Paix et al 2015 ; Dokshin et al 2018 ; Gutierrez-Triana et al 2018 ; Kina et al 2019 ; Wierson et al 2020 ), or treating cells with various cell-cycle drug inhibitors or adenoviral vectors ( Lin et al 2014 ; Gaj et al 2017 ; Zhang et al 2017 ). To our knowledge, no other study has examined Cas9-stimulated HDR targeting of ISG loci, so there is no directly relevant comparison to evaluate the rate of GFP-positive cells with BL3SSO targeting Viperin in HEK293 cells.…”

CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing

“…This suggests that the rate of success in co-editing a GOI has to do with the relative efficiency of the sgRNAs/template for the ts gene and the GOI. Consistent with this, a co-editing system based on HDR-dependent repair of a plasmid-based reporter (HDR-USR) [ 33 ] showed improved yields with more efficient sgRNAs for the plasmid reporter.…”

“…Whether these mutations will impinge on the activity or viability of these cells will need to be checked for the specific conditions and demands of the assay or application. Another approach used repair of a puromycin-resistance gene encoded by a plasmid as a selectable marker [ 33 ]. The advantage of this system is that the selectable gene is expressed transiently on a plasmid.…”

CRISPR-based strategies for targeted transgene knock-in and gene correction

“…Another way to enrich biallelic edited cells is by using a universal surrogate reporter system (HDR-USR) 66 . Compared with the surrogate reporter-integrated donor system 61 , the HDR-USR system functions by itself in an episomal manner 66 . Because the HDR-USR surrogate vector is not integrated into the genome, it allows scarless genome editing without introducing insertional mutagenesis and unwanted exogenous sequences to the genome.…”