SpCas9- and LbCas12a-Mediated DNA Editing Produce Different Gene Knockout Outcomes in Zebrafish Embryos.

Abstract

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) genome editing is a powerful technology widely used in current genetic research. In the most simple and straightforward way it can be applied for a gene knockout resulting from repair errors, induced by dsDNA cleavage by Cas nuclease. For decades, zebrafish (Danio rerio) has been known as a convenient model object of developmental biology. Both commonly used nucleases SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) are extensively used in this model. Among them, LbCas12a is featured with higher specificity and efficiency of homology-directed editing in human cells and mouse. But the editing outcomes for these two nucleases in zebrafish are still not compared quantitatively. Therefore, to reveal possible advantages of one nuclease in comparison to the other in the context of gene knockout generation, we compare here the outcomes of repair of the DNA breaks introduced by these two commonly used nucleases in zebrafish embryos. To address this question, we microinjected the ribonucleoprotein complexes of the both nucleases with the corresponding guide RNAs in zebrafish zygotes and sequenced the target gene regions after three days of development. We found that LbCas12a editing resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies (percentage of mutated copies of the target gene to all gene copies in the embryo) of both nucleases were the same. On the other hand, overlapping of protospacers resulted in similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.