Highly effective gene inactivation in tetraploid Xenopus laevis with low-temperature-active engineered Cas12a

BackgroundGene knockout using the CRISPR/Cas (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein) system revolutionized reverse genetic (or targeted mutagenesis) studies in model and non-model organisms because almost all genetic elements can be targeted with few limitations. Although the CRISPR/Cas system incorporating SpCas9 (Cas9 derived from Streptococcus pyogenes) remains the most popular for genome editing, another CRISPR/Cas system with Cas12a (Cpf1) has expanded its application. However, Cas12a is difficult to use in some aquatic model organisms, such as Xenopus, because of its low activity at the temperature Xenopus is normally raised (lower than 25{degrees}C). Recently, an engineered Cas12a called Cas12a-Ultra was developed, which has improved in vivo endonuclease activity with less temperature dependency, and several commercial products were introduced to the market. ResultsWe evaluated the performance of these engineered Cas12a enzymes in Xenopus embryos. We first confirmed that they were more active than SpCas9 at the low temperature that X. laevis embryos are mostly raised (20-22{degrees}C) based on in vitro digestion experiments. Then, we evaluated their in vivo activities by disrupting the tyrosinase enzyme (tyr), which induces albino-like phenotypes. LbCpf1 (Cpf1 derived from Lachnospiraceae bacterium)-Ultra outperformed the other enzymes, producing more than 80% of embryos with severely defective phenotypes even in low-temperature conditions. In addition, duplicated copies of two paralogous lysine demethylases (kdm5b and kdm5c) were successfully disrupted, which recapitulated the previously reported phenotypes observed upon morpholino-mediated knockdown. ConclusionsNewly engineered Cas12a is valuable for gene function studies in Xenopus and other model organisms with low growth temperatures.