Efficient Gene Disruption via Base Editing Induced Stop in Newt Pleurodeles waltl

Abstract

Loss-of-function approaches provide strong evidence for determining the role of particular genes. The prevalent CRISPR/Cas9 technique is widely used to disrupt target gene with uncontrolled non-homologous end joining after the double strand breaks, which results in mosaicism and multiple genotypes in the founders. In animal models with long generation time such as the salamanders, producing homozygous offspring mutants would be rather labor intensive and time consuming. Here we utilized the base editing technique to create the loss-of-function F0 mutants without the random indels. As a proof of principle, we successfully introduced premature stop codons into the tyrosinase locus and produced the albino phenotype in the newts (Pleurodeles waltl). We further demonstrated that the knockout efficiency could be greatly improved by using multiplex sgRNAs target the same gene. The F0 mutated animals showed fully loss-of-function by both genotyping and phenotyping analysis, which could enable direct functional analysis in the founders and avoid sophisticated breeding. This study not only presented the high efficiency of single base editing in a gigantic animal genome (>20 G), but also provided new tools for interrogating gene function in other salamander species.

Keywords: base editing; genome; newt; sgRNAs.

Figure 1

The scheme of the clustered regularly interspaced short palindromic repeats-associated nucleases (CRISPR)/Cas9 and base editing mediated tyrosinase (tyr) knockout in newt. (A) Five sgRNAs were designed close to the ATG start codon of the tyr exon 1; (B) The genotype and phenotype of the tyr knockout animals produced with the canonical CRISPR/Cas9 technique. The sanger sequencing chromatograms (left panels) and the representative pictures of corresponding animals (right panels) were shown. The predicted protospacer sites were underlined. The NGG pam site was highlighted in blue. The overlapping peaks starting around the PAM site indicated multiple mutant alleles were created. Scale bar: 2 mm. sgRNA (small guide RNA).

Figure 2

The base editing mediated induction of stop codons (iSTOP) in the tyr gene produces albino animals: (A) The genotyping and phenotyping results of the base edited animals. The sequencing chromatograms and the animal pictures showing the base pair conversions created new stop codons (left panels) and varies degrees of albinism (right panels). The asterisks (*) indicate the C-to-T conversions. Scale bar: 2 mm; (B) The alignment of the genomic sequence in the base editing region. Ten TA clones were sequenced from one base edited individual and the frequency of each genotype was shown in the parentheses. The base pair conversions are showing in red. The PAM sites were underlined.

Figure 3

Multiplex sgRNAs could improve the tyr knockout efficiency. The single sgRNAs or multiplex sgRNAs were co-injected with the base editor mRNA into the fertilized eggs and the genotyping and phenotyping were performed 13 days later. The PCR sequencing chromatogram of the three editing sites from the same animal were shown in the left panels. The representative pictures of animals were shown in the right panels. The base pair conversions could be detected in all three editing sites of the individuals with the multiplex sgRNAs injected. Asterisks (*) indicate the base pair conversions. Scale bar: 2 mm.