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

Abstract

Gene knockout using the CRISPR/Cas (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein) system revolutionized reverse genetic studies in model and non-model organisms, as almost all genetic elements can be targeted with few limitations. Although the CRISPR/Cas system with 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 challenging to use in some aquatic model organisms, such as Xenopus laevis, because of its low activity at the temperature at which X. laevis embryos are usually raised (lower than 25{degrees}C). Recently, an engineered Cas12a called Cas12a-Ultra was developed, which has improved in vivo endonuclease activity with reduced temperature dependency. Here, we evaluated the performance of these engineered Cas12a enzymes in X. laevis embryos. We first confirmed that they were more active than SpCas9 at the low temperature at which X. laevis embryos are mostly raised (20-22{degrees}C), based on in vitro digestion experiments. Then, we evaluated in vivo activities of Cas12a-Ultra by disrupting several genes in X. laevis whose phenotypic consequences are previously reported. LbCpf1 (Cas12a 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. This study demonstrated that this engineered Cas12a is valuable for gene function studies in Xenopus and other model organisms with low growth temperatures.