CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Miguel A Moreno-Mateos,Jennifer A Doudna,Antonio J Giraldez,Maura A Lane,Juan P Fernandez,Romain Rouet,Charles E Vejnar,Mustafa K Khokha,Emily Mis

doi:10.1038/s41467-017-01836-2

Abstract

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

Highlights

Cpf[1] is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems
We show that CRISPR-LbCpf[1], together with a single-stranded DNA donor, significantly increases the efficiency of homology-directed repair (HDR) in zebrafish when compared to CRISPR-Cas[9]
To implement Cpf1-mediated genome editing, we compared the activity of recombinant proteins and mRNAs encoding codonoptimized AsCpf[1] or LbCpf[11] injected in one-cell-stage zebrafish embryos

Summary

Introduction

Cpf[1] is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. We show that temperature influences Cpf[1] activity in vivo, affecting AsCpf[1] more markedly, which explains its lower activity in ectothermic organisms such as zebrafish, Xenopus, Drosophila[10], and plants[8, 9, 11] We capitalize on these differences to develop a method that provides temporal control of Cpf1-mediated mutagenesis, resulting in different onset and size of mutant clones. We show that CRISPR-LbCpf[1], together with a single-stranded DNA (ssDNA) donor, significantly increases the efficiency of homology-directed repair (HDR) in zebrafish when compared to CRISPR-Cas[9] All together, these results provide a highly efficient and inducible genome engineering system in ectothermic organisms

Methods

Results

Conclusion