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. 2020 Feb 4;7:10. doi: 10.3389/fmolb.2020.00010

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Copyright © 2020 Arora, Gupta, Vijaykumar and Krishna.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Screening gRNAs for efficient cleavage. (A) in vitro cleavage (IVC) assay for all gRNAs. An MCV template of 2,193 bp spanning the NCCR region to the RB-binding site of T antigen was used for the assay (shown by the arrow). Of the 10 gRNAs tested, six showed efficient cleavage that resulted in visible chopped bands on a 2% agarose gel. (B) Non-specific trans ssDNA shredding activity of AsCas12a-gRNA. M13 DNA phage reporter was subjected to MCV gRNA, complementary ssDNA target (cis-activator) and AsCas12a. All five gRNAs, namely NCCR3, Exon1.1, Exon1.2, Exon2.1, and Exon2.2 resulted in indiscriminate shredding of single-stranded M13 DNA.