Disease modeling by efficient genome editing using a near PAM-less base editor in vivo

Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing the NRN PAM sequence, we could introduce point mutations for the first time in an animal model and achieved up to 100% efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we applied our method to create a new zebrafish model for melanoma predisposition based on the simultaneous editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.

[1]  John R. Garbe,et al.  EditR: A Method to Quantify Base Editing from Sanger Sequencing , 2018, The CRISPR journal.

[2]  Mattie J. Casey,et al.  Pediatric Cancer Models in Zebrafish. , 2020, Trends in cancer.

[3]  M. Firth,et al.  Universal toxin-based selection for precise genome engineering in human cells , 2021, Nature Communications.

[4]  Yves Moreau,et al.  The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models , 2013, Genome Biology.

[5]  Kevin T. Zhao,et al.  Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity , 2017, Science Advances.

[6]  David R. Liu,et al.  Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction , 2018, Nature Biotechnology.

[7]  Martin Distel,et al.  Kita Driven Expression of Oncogenic HRAS Leads to Early Onset and Highly Penetrant Melanoma in Zebrafish , 2010, PloS one.

[8]  J. Bakkers,et al.  Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders , 2018, Disease Models & Mechanisms.

[9]  Iwei Yeh,et al.  Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma , 2018, Science.

[10]  R. Sood,et al.  BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish , 2020, Cells.

[11]  Yihan Zhang,et al.  Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system , 2017, Nature Communications.

[12]  Hong Yan,et al.  Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene , 2015, Nucleic acids research.

[13]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[14]  Sergey V. Prykhozhij,et al.  Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9 , 2018, Nucleic acids research.

[15]  David R. Liu,et al.  Evolved Cas9 variants with broad PAM compatibility and high DNA specificity , 2018, Nature.

[16]  Hanhua Cheng,et al.  An optimized base editor with efficient C-to-T base editing in zebrafish , 2020, BMC biology.

[17]  Jeffry D. Sander,et al.  CRISPR-Cas systems for editing, regulating and targeting genomes , 2014, Nature Biotechnology.

[18]  L. Zon,et al.  Modeling Cancer with Flies and Fish. , 2019, Developmental cell.

[19]  P. Zamore,et al.  Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion , 2016, G3: Genes, Genomes, Genetics.

[20]  Benjamin P. Kleinstiver,et al.  Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants , 2020, Science.

[21]  Carla M. Mann,et al.  Efficient targeted integration directed by short homology in zebrafish and mammalian cells , 2020, eLife.

[22]  Graham Dellaire,et al.  Marker-free coselection for CRISPR-driven genome editing in human cells , 2017, Nature Methods.

[23]  F. Del Bene,et al.  Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish , 2020, bioRxiv.

[24]  F. V. van Eeden,et al.  The Zebrafish as an Emerging Model to Study DNA Damage in Aging, Cancer and Other Diseases , 2019, Front. Cell Dev. Biol..

[25]  Kevin T. Zhao,et al.  Improved base excision repair inhibition and bacteriophage , 2017 .

[26]  Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ) , 2018, Disease Models & Mechanisms.

[27]  L. Zon,et al.  Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish. , 2009, Zebrafish.

[28]  C. Mello,et al.  A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis elegans , 2014, Genetics.

[29]  David R. Liu,et al.  Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage , 2016, Nature.