ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

SIMPLE SUMMARY: CRISPR/Cas9 enables efficient mutagenesis and generation of various knockout and knockin alleles in many species including zebrafish. However, the application of the Cas12a nuclease in zebrafish is far from ideal due to demanding experimental conditions, especially the requirements for delivery such as a purified protein and the heatshock of embryos. Here we show that ErCas12a, the only Cas12a reported to be effective when injected as mRNA in zebrafish, is highly efficient for large fragment knockin via either microhomology-mediated or non-homologous end joining pathways with mild heatshock conditions. Moreover, we fused T5 exonuclease to ErCas12a and found that the fusion protein could efficiently induce gene knockout and knockin without heatshock. Therefore, we demonstrated the efficacy of multiple genome-editing applications using ErCas12a and its variant with simplified conditions in zebrafish. ABSTRACT: In zebrafish, RNA-guided endonucleases such as Cas9 have enabled straightforward gene knockout and the construction of reporter lines or conditional alleles via targeted knockin strategies. However, the performance of another commonly used CRISPR system, Cas12a, is significantly limited due to both the requirement of delivery as purified protein and the necessity of heatshock of injected embryos. To explore the potential of CRISPR/Cas12a-mediated genome editing and simplify its application in zebrafish, we took advantage of the recently reported mRNA-active ErCas12a and investigated its efficacy for the knockin of large DNA fragments, such as fluorescent reporter genes. For knockin via either microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ) pathways, ErCas12a-injected embryos with a brief heatshock displayed comparable knockin efficiency with Cas9 injection. Through the fusion of T5 exonuclease (T5exo) to the N-terminus of ErCas12a (T5exo-ErCas12a), we further demonstrated high efficiency gene knockout and knockin at a normal incubation temperature, eliminating the embryo-damaging heatshock step. In summary, our results demonstrate the feasibility of ErCas12a- and T5exo-ErCas12a-mediated genome manipulation under simplified conditions, and further expand the genome editing toolbox for various applications in zebrafish.