Stem-loop adapter, polynucleotide library for grna production, and methods for using same

The stem-loop adapter and PCR-based method efficiently produce gRNA libraries from small amounts of genome material, addressing the inefficiencies of current methods by enabling rapid and cost-effective genome and epigenome editing, including non-coding region analysis.

WO2026071097A1 Publication Date: 2026-04-02YOSHIDA KEISUKE +2
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Patent Information

Application Number
PCT/JP2025/034153
Authority / Receiving Office
WO · WO
Patent Type
Applications
Current Assignee / Owner
Priority Date
2024-09-27
Filing Date
2025-09-26
Publication Date
2026-04-02

AI Technical Summary

Technical Problem

Current methods for producing gRNA libraries are costly, time-consuming, and inefficient, particularly when dealing with small amounts of genome material, and do not effectively address the need for comprehensive genome editing and phenotypic analysis.

Method used

A stem-loop type adapter is combined with PCR technology to produce a polynucleotide library for gRNA production, utilizing adapters A and B to facilitate rapid, simple, and inexpensive generation of gRNA libraries from small amounts of polynucleotide fragments.

Benefits of technology

Enables the easy, rapid, and cost-effective production of diverse gRNA libraries spanning the entire target genome, allowing for comprehensive genome and epigenome editing, including analysis of non-coding regions and identification of gene mutations associated with phenotypic changes.

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Abstract

[Problem] To provide: an adapter for binding to polynucleotide fragments that is used for preparing a genome-wide gRNA library for genome editing systems; a gRNA library that is obtained by using the adapter; and uses of the gRNA library. [Solution] An adapter having a structure described in A). A) A structure having i) to iii). i) A polynucleotide chain having sequences i)-1 to i)-3. i)-1: Sequence corresponding to the forward primer. i)-2: Sequence of the recognition site of a restriction enzyme capable of excising a gRNA spacer sequence from the 5' end of a polynucleotide fragment bound to the adapter. i)-3: Sequence of the recognition site of a restriction enzyme capable of cleaving an adapter having the structure of A) from the polynucleotide fragment. ii) A polynucleotide chain having the sequence complementary to i). iii) A single-stranded polynucleotide chain for formation of a loop connecting the 5' end of i) and the 3' end of ii) of the double strand resulting from the binding of i) and ii).
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