Highly effective gene inactivation in tetraploidXenopus laeviswith low-temperature-active engineered Cas12a

Abstract

AbstractBackgroundGene 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 fromStreptococcus 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 asXenopus, because of its low activity at the temperatureXenopusis normally raised (lower than 25°C). Recently, an engineered Cas12a called Cas12a-Ultra was developed, which has improvedin vivoendonuclease activity with less temperature dependency, and several commercial products were introduced to the market.ResultsWe evaluated the performance of these engineered Cas12a enzymes inXenopusembryos. We first confirmed that they were more active than SpCas9 at the low temperature thatX. laevisembryos are mostly raised (20–22°C) based onin vitrodigestion experiments. Then, we evaluated theirin vivoactivities by disrupting the tyrosinase enzyme (tyr), which induces albino-like phenotypes. LbCpf1 (Cpf1 derived fromLachnospiraceaebacterium)-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 (kdm5bandkdm5c) were successfully disrupted, which recapitulated the previously reported phenotypes observed upon morpholino-mediated knockdown.ConclusionsNewly engineered Cas12a is valuable for gene function studies inXenopusand other model organisms with low growth temperatures.