Efficient Homology-directed Repair with Circular ssDNA Donors

摘要: While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies cell types, well integration at unintended sites. In contrast, single-stranded (ssDNA) can produce efficient with minimal off-target integration. Here, we describe use ssDNA phage to efficiently and inexpensively long circular (cssDNA) donors. These cssDNA serve templates when Cas9 or Cas12a, frequencies superior linear (lssDNA) To evaluate relative imprecise precise for a suite different Cas12a have developed modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons nuclease systems. We this assess platforms distinct donor types. then extended analysis types fluorescent tag knock-ins endogenous sites HEK293T K562 cells. Our results show robust insertion reporter tags. Targeting efficiency is high, allowing production biallelic integrants using also outcompete lssDNA template-driven target site. data demonstrate provide an cost-effective method achieve mammalian lines.