Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking

Md Lutfur Rahman,Hiroyuki Konishi,Yuko Mihara,Md Wahiduzzaman,Sivasundaram Karnan,Shinobu Tsuzuki,Akinobu Ota,Yoshitaka Hosokawa,Muhammad Nazmul Hasan,Toshinori Hyodo

doi:10.1038/s41598-021-01978-w

Md Lutfur Rahman, Hiroyuki Konishi + Show 8 more

Open Access

https://doi.org/10.1038/s41598-021-01978-w

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Abstract

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700–2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.

Highlights

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response
One methodology aligned with the nicking-based strategy for targeted knock-in is tandem paired nicking[3,4,5], in which the target sites of two Cas[9] nickases are placed on the region of homology between the donor DNA and the genome in such a way that the target sequences of two Cas[9] nickases encompass the knock-in site and the nicks are introduced in the same DNA strand (Supplementary Fig. S1)
Our previous study demonstrated that the length of homology between donor DNAs and the genome affects the efficiency of targeted knock-in significantly more in TPN than it does in the Cas[9] nuclease-based approach[5]

Summary

Introduction

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. The use of TPN can be advantageous for the editing of coding genes because transcription, splicing, translation, and degradation of mRNAs originating from edited genes are potentially affected by the genomic incorporation of additional nucleotide changes, even when they are silent As another advantage of TPN-based targeted knock-in, Cas[9] nickases designed against genomic sites fairly distant from the knock-in sites (up to 416-bp distant in CD55 gene targeting), albeit within the region of homology, are capable of promoting relatively efficient targeted knock-in[5]. This flexibility may pose a challenge when attempting to identify an optimal pair of Cas[9] nickases with which to conduct TPN-based targeted knock-in with the highest achievable efficiency, because guidelines for choosing optimal Cas[9] nickases for TPN are currently unavailable

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Conclusion