RT Journal Article
SR Electronic
T1 A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.05.11.088252
DO 10.1101/2020.05.11.088252
A1 Eric Danner
A1 Mikhail Lebedin
A1 Kathrin de la Rosa
A1 Ralf Kühn
YR 2020
UL http://biorxiv.org/content/early/2020/05/12/2020.05.11.088252.abstract
AB Precision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.Competing Interest StatementE.D and R.K. have filed a patent on this subject (WO2019/122302A1).