Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

Yaoge Jiao,Li Li,Lurong Jiang,Yun Hu,Lifang Zhou,Yanhong Wang,Rui Tao,Shaohua Yao

doi:10.1186/s43556-021-00057-w

Abstract

Prime editing (PE) enables efficiently targeted introduction of multiple types of small-sized genetic change without requiring double-strand breaks or donor templates. Here we designed a simple strategy to introduce random DNA sequences into targeted genomic loci by prime editing, which we named random prime editing (Random-PE). In our strategy, the prime editing guide RNA (pegRNA) was engineered to harbor random sequences between the primer binding sequence (PBS) and homologous arm (HA) of the reverse transcriptase templates. With these pegRNAs, we achieved efficient targeted insertion or substitution of random sequences with different lengths, ranging from 5 to 10, in mammalian cells. Importantly, the diversity of inserted sequences is well preserved. By fine-tuning the design of random sequences, we were able to make simultaneously insertions or substitutions of random sequences in multiple sites, allowing in situ evolution of multiple positions in a given protein. Therefore, these results provide a framework for targeted integration of random sequences into genomes, which can be redirected for manifold applications, such as in situ protospacer adjacent motif (PAM) library construction, enhancer screening, and DNA barcoding.

Highlights

The genome editing tools based on Clustered regularly interspaced palindromic repeats (CRISPR) Cas9 have shown significant successes in basic biomedical research and provided great promise in clinical translation [1,2,3]
In vivo gene evolution via random‐Prime editing (PE) After establishing that our Random-PE was efficient in introducing random sequences into the mammalian genome, we further examined if Random-PE could be rewired for the purpose of unbiased gene evolution [13, 14]
We showed that Random-PE achieved targeted integration of up to 10 bp random sequence at an efficiency of up to 39.07%

Summary

Introduction

The genome editing tools based on Clustered regularly interspaced palindromic repeats (CRISPR) Cas have shown significant successes in basic biomedical research and provided great promise in clinical translation [1,2,3]. The development of prime editing technique enables targeted introduction of multiple types of small-sized genetic change in the genome, including deletion, insertion, and base substitution, in an efficient and irreversible way [4]. This technique does not require the generation of double-strand breaks within the target site, nor does it require donor templates [4]. PegRNA is the soul of the PE system in that it guides the Cas and RT fusion protein (PE2) to the target site to produce a nick in the edited strand, and provides the nicked DNA with primer binding sequence (PBS) and RT template for the reverse transcription of the former [4]. By fine-tuning the design of pegRNA, prime editing can achieve really rewriting the genome

Methods

Results

Conclusion