Efficient Homology-directed Repair with Circular ssDNA Donors

Abstract

32While genome editing has been revolutionized by the advent of CRISPR-based 33 nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair 34(HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer 35 from low HDR efficiencies in many cell types, as well as integration at unintended sites. 36In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with 37 minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently 38 and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors 39 serve as efficient HDR templates when used with Cas9 or Cas12a, with integration 40 frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative 41 efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a 42 nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-43

Precision Cas9 Genome Editingin vivowith All-in-one, Self-targeting AAV Vectors

“…To resolve this issue, we used λred phage recombineering technology 78 to integrate the H. parainfluenzae anti-CRISPR acrIIc4 Hpa gene 79 , under the control of its native promoter, into DH5α competent E. coli cells to inhibit Nme2Cas9 and prevent plasmid degradation. These plasmids were tested in a Traffic Light Reporter (TLR) system 80 , TLR-Multi-Cas-Variant 1 (MCV1), that has a disrupted GFP coding sequence followed by an out-of-frame mCherry cassette 81 . The reporter cassette was inserted as a single copy via lentiviral transduction in HEK293T cells.…”

“…HEK293T cells harboring Traffic Light Reporter Multi-Cas Variant 1 (TLR-MCV1) 81 , as well as Neuro2a cells (ATCC CCL-131), were cultured in Dulbecco’s Modified Eagle Media (DMEM, Thermo Fisher Scientific, Cat. No.…”

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

“…Finally, we tested the efficiency for generating a targeted insertion using PE2, PE2* and Sa KKH PE2* in a different HEK293T reporter line (TLR-MCV1) that can quantify both precise insertions and indel formation 23 . A targeted, precise replacement of 39bp disruption sequence with a 18bp missing sequence element can restore GFP expression, whereas indels that produce a different reading frame alteration restore mCherry expression ( Supplementary Fig.…”

“…reporter cells was described in 23 . All cell types were maintained at 37°C and 5% CO2 and were tested negative for mycoplasma.…”

“…Two weeks post selection, hygromycin resistant foci were pooled and propagated for cryopreservation and further experiments. The construction an characterization of the TLR-MCV1 reporter cells was described in 23 . All cell types were maintained at 37°C and 5% CO 2 and were tested negative for mycoplasma.…”