PT  - JOURNAL ARTICLE
AU  - Bin Zhao
AU  - Shi-An A. Chen
AU  - Jiwoo Lee
AU  - Hunter B. Fraser
TI  - Bacterial retrons enable precise gene editing in human cells
AID  - 10.1101/2021.03.29.437260
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.03.29.437260
4099  - http://biorxiv.org/content/early/2021/03/29/2021.03.29.437260.short
4100  - http://biorxiv.org/content/early/2021/03/29/2021.03.29.437260.full
AB  - Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve editing efficiency of precise genome editing via homology-directed repair (HDR). HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.3%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.Competing Interest StatementStanford University has applied for a patent on the CRISPEY method.