High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs.

Duda K, Lonowski LA, Kofoed-Nielsen M, Ibarra A, Delay CM, Kang Q, Yang Z, Pruett-Miller SM, Bennett EP3, Wandall HH, Davis GD, Hansen SH, Frödin M.
Source: Nucleic Acids Res
Publication Date: (2014)
Issue: 42: 1-16
Research Area:
Cancer Research/Cell Biology
Gene Expression
Cells used in publication:
Species: human
Tissue Origin: blood
Species: human
Tissue Origin: breast
Species: human
Tissue Origin: blood
HCT 116
Species: human
Tissue Origin: colon
Nucleofector® I/II/2b
Transfection of HCT-116: For mRNA transfection, HCT116 cells were split and plated at a density leading to ~70% confluency at the time of transfection 2 days later. Cells were then electroporated using a Nucleofector (Lonza) set to program D-032. 1 × 106 cells were dissolved in 100-µl Nucleofector Solution V (Lonza) with 2.0-µg and 3.3µg mRNA transcribed from pZFN and pFluo-2A-ZFN, respectively. The difference in mRNA amounts used was due to the size difference of the mRNA constructs (~1170 and ~1950 bps, respectively, plus a poly A tail of ~150 bps) and ensured transfection with roughly equimolar amounts of both mRNA types. Transfection of K562: The K562 electroporation was performed in a Nucleofector (Lonza) using program T-016 according to the manufacturer’s instructions. Briefly, 1 × 106 cells in 100-µl Nucleofector Solution V (Lonza) were mixed with the following reagents as appropriate for the experiment: for ssODN donor-based genome editing, 3 µl of 100-µM RSK4 Cys443Val/BamHI ssODN donor (equaling ~6.7 µg) in 10-mM Tris (pH 7.6) was used together with 2-µg ZFN-expressing plasmid (for each ZFN of the pair) or with 2-µg and 3.3-µg ZFN-expressing mRNA (for each ZFN of the pair), when transcribed from pZFN and pFluo-2A-ZFN, respectively. For plasmid donor-based genome editing, 10-µg RSK2 Cys436Val/BamHI plasmid donor was used together with 2-µg ZFN-expressing mRNA when transcribed from pZFN or 3.3-µg ZFN-expressing mRNA when transcribed from pFluo-2A-ZFN. Transfection of MCF-7 and Jurkat: MCF10A and Jurkat cells were electroporated as described for K562 except that Nucleofector solution T/Nucleofector programT-020 and Nucleofector Solution V/Nucleofector program X-001 were used for the two cell lines, respectively. In the comparison of the efficiency of ZFN genome editing using non-linked versus 2Alinked fluorescent protein, the non-linked fluorescent protein was delivered as 250 ng of pGFP plasmid (contained in the Nucleofector kit from Lonza).
Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and ~ 2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ~ 1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.