RT Journal Article
SR Electronic
T1 Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.11.01.466790
DO 10.1101/2021.11.01.466790
A1 Andrew V. Anzalone
A1 Xin D. Gao
A1 Christopher J. Podracky
A1 Andrew T. Nelson
A1 Luke W. Koblan
A1 Aditya Raguram
A1 Jonathan M. Levy
A1 Jaron A. M. Mercer
A1 David R. Liu
YR 2021
UL http://biorxiv.org/content/early/2021/11/02/2021.11.01.466790.abstract
AB The targeted deletion, replacement, integration, or inversion of DNA sequences at specified locations in the genome could be used to study or treat many human genetic diseases. Here, we describe twin prime editing (twinPE), a method for the programmable replacement or excision of DNA sequence at endogenous human genomic sites without requiring double-strand DNA breaks. TwinPE uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences. We show that twinPE in human cells can perform precise deletions of at least 780 bp and precise replacements of genomic DNA sequence with new sequences of at least 108 bp. By combining single or multiplexed twinPE with site-specific serine recombinases either in separate steps or in a single step, we demonstrate targeted integration of gene-sized DNA plasmids (>5,000 bp) into safe-harbor loci including AAVS1, CCR5, and ALB in human cells. To our knowledge, these results represent the first RNA-programmable insertion of gene-sized DNA sequences into targeted genomic sites of unmodified human cells without requiring double-strand breaks or homology-directed repair. Twin PE combined with recombinases also mediated a 40,167-bp inversion at IDS that corrects a common Hunter syndrome allele. TwinPE expands the capabilities of precision gene editing without requiring double-strand DNA breaks and synergizes with other tools to enable the correction or complementation of large or complex pathogenic alleles in human cells.Competing Interest StatementD.R.L. is a consultant and equity holder of Beam Therapeutics, Prime Medicine, Pairwise Plants, and Chroma Medicine, companies that use genome editing or genome engineering technologies. A.V.A., C.J.P., and J.M.L. are currently employees at Prime Medicine. The authors have filed patent applications on twinPE and prime editing through the Broad Institute.