Expanding The CRISPR Toolbox With Mad7 In Zebrafish And Human Cells

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) effector proteins enable the direction of DNA double-strand breaks at defined loci based on a variable length RNA guide specific to each effector. The guides are generally similar in size and form, consisting of a ~20 base sequence homologous to the DNA target and a secondary structure used by the effector for guide/nuclease recognition. However, the effector proteins vary in size, DNA binding kinetics, nucleic acid hydrolyzing activities, and Protospacer Adjacent Motif (PAM) requirements. Recently, a Cas12a family member protein named Mad7 was identified that is most similar to the Cas12a from Acidaminococcus sp. Here, we report for the first time Mad7 activity in zebrafish and human cells. We utilize a fluorescent reporter system to demonstrate CRISPR/Mad7 elicits strand annealing mediated DNA repair more efficiently than CRISPR/Cas9. Finally, we use CRISPR/Mad7 with our previously reported gene targeting method GeneWeld in order to integrate reporter alleles in both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR/Cas system, increasing the flexibility researchers have in applying genome engineering technologies.