Abstract
Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. While CRISPR-based gene editing of multiple genes at once offers an attractive alternative strategy, subsequent breeding or establishment of permanent mouse lines will rapidly segregate the different mutant loci again. Direct phenotypic analysis of genomic edits in an embryonic lethal gene in F0 generation mice, or F0 mouse embryos, circumvents the need for breeding or establishment of mutant mouse lines. In the course of genotyping a large cohort of F0 CRISPants, where the embryonic lethal gene T/brachyury was targeted, we noted the presence of multiple CRISPR-induced modifications in individual embryos. Using long-read single-molecule Nanopore sequencing, we identified a wide variety of deletions, ranging up to 3 kb, that would not have been detected or scored as wildtype with commonly used genotyping methods that rely on subcloning and short-read or Sanger sequencing. Long-read sequencing results were crucial for accurate genotype–phenotype correlation in our F0 CRISPants. We thus demonstrate feasibility of screening manipulated F0 embryos for mid-gestation phenotypic consequences of CRISPR-induced mutations without requiring derivation of permanent mouse lines.
Highlights
Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse
We here report that similar morphological anomalies were observed in mouse embryos where the T/brachyury gene was edited by CRISPR mutagenesis, carried out in a transient transgenic strategy that involves phenotyping and genotyping of individuals directly in the F0 generation
Our goal in this work was to test the feasibility of a transient approach to identify new genes that—alone or in combination—contribute to morphogenesis and closure of the neural tube
Summary
Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. The approach is called transient, as the phenotype resulting from the genetic manipulation is analyzed directly in the F0 generation, without establishment of permanent mouse lines[3,4] The advantage of such a strategy is that it allows for the screening of genes whose bi-allelic disruption may cause embryonic lethality, precluding the derivation of permanent mouse lines[5]. We here report that similar morphological anomalies were observed in mouse embryos where the T/brachyury gene was edited by CRISPR mutagenesis, carried out in a transient transgenic strategy that involves phenotyping and genotyping of individuals directly in the F0 generation It was only by employing single-molecule long-read sequencing, as implemented in the Oxford Nanopore sequencing technology[14], that we were able to detect the multiple CRISPR-edited mutant T/brachyury alleles in single F0 individuals, and to identify larger on-target deletions than have been reported from previous CRISPR mutagenesis efforts
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