Efficient SSA‐mediated precise genome editing using CRISPR/Cas9

Abstract

CRISPR/Cas9 has been emerging as a main player in genome editing field since its advent. However, CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing. Among the few reports regarding two-step 'pop in & out' technologies for precise gene editing, the combination of CRISPR/Cas9 with Cre/LoxP demonstrates a higher efficiency, but leaves behind a 34-base pair of tag sequence due to its inherent property. Another method utilizes piggyBac transposon for removing the selection cassette, and its disadvantage is the difficulty in controlling its random reintegration after releasing. Here, we report a novel two-step precise gene-editing method by leveraging the SSA-mediated repair mechanism into the CRISPR/Cas9-mediated gene-editing system. An integrating cassette was developed with positive and negative selection markers, which was flanked by direct repeat sequences with desired mutations as SSA arms. After the targeted integration of the cassette mediated by CRISPR/Cas9-induced homologous-directed repair, cell clones were first selected through the positive selection. In the second round targeting, the selection cassette was removed by the SSA-mediated DNA double-strand break (DSB) repair without any scar left behind. The novel seamless genome editing technique was tested on CCR5 and APP loci, and finally demonstrated, respectively, up to 45.83% and 68% of precise genome editing efficiency. This study provides a new efficient approach for precise genome editing and gene correction.

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

“…Other forms of modifications, such as large deletions and inversions, can also be achieved using optimized endonucleases. In addition to NHEJ and HDR, a single-strand annealing (SSA) repair mechanism can also be utilized to mediate modifications by inducing the deletion of an intervening fragment between two homogenous repeat sequences (Li et al, 2018c).…”

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

“…This is subsequently followed by a second step that results in the excision of a marker from the knockin cassette during precision editing of the target locus, allowing enrichment by negative selection. 32,33 Importantly, a recent report demonstrated that HDR-dependent insertion of a selectable marker at one locus coincides with one or more independent HDR-mediated edits at other loci. The concept forms the basis for a strategy has been termed "co-targeting with selection," which can be used to efficiently enrich for HDR-proficient cells.…”

Editorial: Precise Genome Editing Techniques and Applications

“…However, the removal of the selection is often required to allay concerns of marker-dependent effects. There are several "pop in and out" two-step techniques for markerfree genome engineering, including the Cre/LoxP system (Zhu et al, 2015), the piggyBac transposon (Xie et al, 2014), and the SSA repair mechanism (Li et al, 2018). This Research Topic presents a protocol article for biallelic HDR targeting using piggyBac-mediated selection removal (Jarazo et al).…”