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

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...

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Autores principales:	Yaoge Jiao, Lifang Zhou, Rui Tao, Yanhong Wang, Yun Hu, Lurong Jiang, Li Li, Shaohua Yao
Formato:	article
Lenguaje:	EN
Publicado:	Springer 2021
Materias:	CRISPR/Cas9 Prime editing Random sequence Gene evolution Medicine R
Acceso en línea:	https://doaj.org/article/d5b86a6e19364c48be43fc0880890585
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spelling	oai:doaj.org-article:d5b86a6e19364c48be43fc08808905852021-11-21T12:03:47ZRandom-PE: an efficient integration of random sequences into mammalian genome by prime editing10.1186/s43556-021-00057-w2662-8651https://doaj.org/article/d5b86a6e19364c48be43fc08808905852021-11-01T00:00:00Zhttps://doi.org/10.1186/s43556-021-00057-whttps://doaj.org/toc/2662-8651Abstract 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.Yaoge JiaoLifang ZhouRui TaoYanhong WangYun HuLurong JiangLi LiShaohua YaoSpringerarticleCRISPR/Cas9Prime editingRandom sequenceGene evolutionMedicineRENMolecular Biomedicine, Vol 2, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR/Cas9 Prime editing Random sequence Gene evolution Medicine R
spellingShingle	CRISPR/Cas9 Prime editing Random sequence Gene evolution Medicine R Yaoge Jiao Lifang Zhou Rui Tao Yanhong Wang Yun Hu Lurong Jiang Li Li Shaohua Yao Random-PE: an efficient integration of random sequences into mammalian genome by prime editing
description	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.
format	article
author	Yaoge Jiao Lifang Zhou Rui Tao Yanhong Wang Yun Hu Lurong Jiang Li Li Shaohua Yao
author_facet	Yaoge Jiao Lifang Zhou Rui Tao Yanhong Wang Yun Hu Lurong Jiang Li Li Shaohua Yao
author_sort	Yaoge Jiao
title	Random-PE: an efficient integration of random sequences into mammalian genome by prime editing
title_short	Random-PE: an efficient integration of random sequences into mammalian genome by prime editing
title_full	Random-PE: an efficient integration of random sequences into mammalian genome by prime editing
title_fullStr	Random-PE: an efficient integration of random sequences into mammalian genome by prime editing
title_full_unstemmed	Random-PE: an efficient integration of random sequences into mammalian genome by prime editing
title_sort	random-pe: an efficient integration of random sequences into mammalian genome by prime editing
publisher	Springer
publishDate	2021
url	https://doaj.org/article/d5b86a6e19364c48be43fc0880890585
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Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

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