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. 2019 Mar 13;11(3):255. doi: 10.3390/v11030255

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© 2019 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Sequence of the target region upon breakthrough virus replication. (a–b) When virus replication was observed in dual-gRNA expressing Jurkat cells (gGag1 + gEnv2 cells in a; gGag1+gTatRev cells in b), the viral target sequence was analyzed. The sequence fully complementary to the gRNA and the PAM (in bold), as present in the LAI isolate, are shown on top. The Cas9 cleavage site is indicated with a red arrow. For each isolate, the target sequence of the breakthrough virus is shown, with pre-existing gRNA-mismatching nucleotides indicated in red and newly acquired mutations observed upon replication indicated in green. The amino acid sequence of the encoded proteins is shown with the pre-existing and acquired amino acid differences indicated in red and green, respectively.