Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Abstract

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient biallelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

Early frameshift alleles of zebrafish <em>tbx5a</em> that fail to develop the heartstrings phenotype

“…We readily observed this phenotype using translation-blocking tbx5a morpholino injections (n=30/106) (Fig. 1D), in line with previous reports of variable expressivity [7] [19] [11]. The presence of the hst phenotype itself has previously also been linked to the genetic background [7], suggesting that the hst phenotype is a variation of the tbx5a loss-of-function phenotype.…”

“…1A) [11]. This sgRNA targets the coding sequence in the first coding exon downstream of the conserved translation initiation codon [12].…”

“…Further indicating that targeting this region could result in loss-of-function alleles, the corresponding amino acid sequence is highly conserved between zebrafish and humans (indicating functional conservation) and human HOS patients have been identified with frameshift-introducing nucleotide insertions at similar positions within TBX5 [13]. Maximized mutagenesis using Cas9 RNPs with sgRNA[tbx5ccA] cause recognizable tbx5a loss-of-function phenotypes in F0 crispants [11]. We injected the sgRNA complexed with Cas9 protein as solubilized RNPs [11] at sub-optimal concentration to achieve viable mosaicism (see Methods for details) in the multicolor Tg(lmo2:dsRED2;drl:EGFP;myl7:mCyan) reporter background, subsequently abbreviated as RGB.…”

“…Maximized mutagenesis using Cas9 RNPs with sgRNA[tbx5ccA] cause recognizable tbx5a loss-of-function phenotypes in F0 crispants [11]. We injected the sgRNA complexed with Cas9 protein as solubilized RNPs [11] at sub-optimal concentration to achieve viable mosaicism (see Methods for details) in the multicolor Tg(lmo2:dsRED2;drl:EGFP;myl7:mCyan) reporter background, subsequently abbreviated as RGB. In RGB embryos, dsRED2 labels endothelial, hematopoietic, and endocardial progenitors (lmo2) in red [14], EGFP marks all lateral plate mesoderm lineages (drl) including pectoral fins in green [15], and AmCyan reveals the differentiated cardiomyocytes (myl7 ) in blue [16]; consequently, RGB enables in vivo imaging of all cardiovascular and additional LPM lineages over the first 3 days of development.…”

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

“…In zebrafish, mutagenesis can be performed through microinjection of Cas9-encoding mRNA or of Cas9 protein together with sgRNA into fertilized embryos. Contrary to Cas9-encoding mRNA, RNPs are immediately active upon microinjection and are generally more effective in the fast dividing blastomeres of the zebrafish and Xenopus embryos (Figure 1) [37,38], in which mutagenesis occurring after the first cleavages leads to increased mosaicism. In plants, this DNA-free approach allows generation of marker-free ‘cisgenic’ variants that could be exempted from current GMO regulations and thereby supersede all the technologies of gene editing in plants [39].…”