Broad spectrum of CRISPR-induced edits in an embryonic lethal gene

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.

Correspondence of yolk sac and embryonic genotypes in F0 mouse CRISPants

“…These phenotypes are also observed in our newly generated independent F0 CRISPants, which recapitulate some gene editing events identified in our previous study 11 , but also contained distinct new alleles. Moreover, we failed to detect unique mutant alleles in the embryo that weren’t also present in the yolk sac.…”

“…This guide RNA targets exon 3, which encodes a part of the DNA-binding domain of the T transcription factor. We showed that disruptions of this exon by CRISPR-induced deletions produce similar morphological anomalies as observed in conventional T mutant embryos 11 : a wide variety of defects in mesoderm derivatives, tails that are shorter, curled or absent, abnormal neural tube closure, kinked or wavy neural tubes 15 .…”

“…In approaches designed to rely on the repair of CRISPR/Cas9-induced DNA double strand breaks by non-homologous end-joining (NHEJ), small deletions of up to 10 base pairs can be expected at the target cut site theta 10 . However, we previously discovered that a single guide RNA targeted to the mouse T/brachyury gene, when co-injected with Cas9 protein into fertilized mouse oocytes, can generate numerous deletion variants with breakpoints even far away from the target site theta, and of varying length and direction 11 .…”

“…In addition, we detected multiple variant alleles within the same animal, implying that the first Cas9-guideRNA-induced DNA break occurred only on one chromosome, and that additional mutations were induced during further cell division(s). Since any diploid cell can only contain two alleles of any given gene, any individual with 3 or more distinct alleles has to be a mosaic, composed of cells that contain distinct CRISPR induced mutant alleles 11 .…”

“…In the present study, we conducted explicit tests for this possibility, by genotyping both embryo and yolk sac from the same conceptus at midgestation, using a validated guide RNA directed to the same target site in the T/brachyury locus, published previously 11 . This guide RNA targets exon 3, which encodes a part of the DNA-binding domain of the T transcription factor.…”