bioRxivGenomics
Easi-CRISPR: Efficient germline modification with long ssDNA donors
Rolen M Quadros, Masato OhtsukaiD, Donald W Harms, Tomomi Aida, Ronald Redder, Hiromi MiuraiD, Guy P Richardson, Mark A Behlke, Sarah A Zeiner, Ashley M Jacobi, Lisa D Urness, Suzanne L Mansour, Channabasavaiah B Gurumurthy
Abstract
CRISPR/Cas9 technology efficiently produces short insertions or deletions (indels) and can insert short exogenous sequences at Cas9 cut sites. However, targeting long inserts is still a major technical challenge. To overcome this challenge, we developed Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a method that uses long, in vitro-synthesized, single-stranded DNAs with 50-100 base homology arms as repair templates. We demonstrate that Easi-CRISPR can generate knock-in and floxed alleles in mice with an efficiency at many loci as high as 100%. The simple design requirements for donor DNAs and the reproducibly high-efficiency of Easi-CRISPR enables rapid development of many types of commonly used animal and cell models.
- organism: mouse
- assay: CRISPR screen
- research focus: perturbation response prediction
- approach: experimental assay
Reproducibility ledger
Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.
Sign in to file a reproducibility report.
Related papers
- An Efficient CRISPR Protocol For Generating Conditional And Knock-In Mice Using Long Single-Stranded DNA Donors
Hiromi Miura, Rolen M. Quadros, … Masato Ohtsuka·Nature ProtocolsPeer-reviewed·Dec 21, 2017
- Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans
Matthew Eroglu, Bin Yu, W. Brent Derry·FEBS JournalPeer-reviewed·May 31, 2023
- New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing
Dorjee T.N. Shola, Chingwen Yang, … Victor Bustos·The CRISPR JournalPeer-reviewed·Apr 1, 2020
- An Improved Protocol for High-Efficiency and Cost-Effective CRISPR/Cas9-Mediated Knock-ins in C. elegans
Atal Vats, Arunima Sen, … Abhishek Bhattacharya·bioRxiv·Sep 7, 2026
- Design and specificity of long ssDNA donors for CRISPR-based knock-in
Han Li, Kyle A. Beckman, … Manuel D. Leonetti·bioRxiv·Nov 20, 2019
- An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms
Oguz Kanca, Jonathan D. Zirin, … Hugo Jozef Bellen·eLifePeer-reviewed·Nov 1, 2019