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Differential nicking patterns of Cas12a variants
Author(s) -
Murugan Karthik,
Sashital Dipali G
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.620.7
Subject(s) - cas9 , biology , endonuclease , crispr , cleavage (geology) , genetics , computational biology , genome editing , trans activating crrna , microbiology and biotechnology , gene , paleontology , fracture (geology)
Cas12a is an endonuclease from the bacterial CRISPR‐Cas immune system that can be easily programmed for use in various biotechnological applications. Despite its prevalent use in genome editing, the cleavage specificity and activity of Cas12a remain to be fully determined. We employed high‐throughput in vitro cleavage assays to determine and compare the native specificities of different Cas12a variants. Surprisingly, we observe pervasive nicking of randomized target libraries. Cas12a has distinct nicking activity against targets with multiple mismatches, where it can linearize certain mismatch targets, but only nick others. Our results also demonstrate that Cas12a variants have differential in vitro cleavage activity and mismatch tolerance. Francisella novicida (Fn)Cas12a is less tolerant to mismatches than Lachnospiraceae bacterium (Lb)Cas12a and Acidaminococcus sp (As)Cas12a. The mismatch position plays a vital role in determining target specificity of Cas12a. Like Streptococcus pyogenes (Sp)Cas9, the mismatches in the PAM‐proximal “seed” region are especially deleterious to Cas12a target cleavage, however, the seed region is shorter (~ 6bp) when compared to SpCas9 (~ 8–10bp). Overall, we observe that Cas12a variants have sequence‐ and seed‐specific nicking and cleavage activities, with variable sensitivities to mismatch position. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .