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. 2020 Mar 25;21:78. doi: 10.1186/s13059-020-01989-2

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — CeCas12a and BfCas12a mediate gene editing in human cells. a Efficiencies of EGFP disruption in human cells mediated by CeCas12a and crRNAs bearing variable-length complementarity regions for the target site of EGFP in human cells. Error bars indicate standard errors of means (s.e.m.), n = 3. ***P < 0.001 (Mann-Whitney). b Efficiencies of EGFP disruption mediated by BfCas12a and crRNAs bearing variable-length complementarity regions for the target site of EGFP in human cells. Error bars represent s.e.m., n = 3. ***P < 0.001 (Mann-Whitney). c Efficiencies of EGFP disruption in human cells mediated by Cas12a orthologs and crRNA bearing 23-nt length complementarity regions for the target site of EGFP in human cells. Error bars represent s.e.m., n = 3. ***P < 0.001 (Mann-Whitney). d The suitability of Cas12a with crRNAs containing different loop regions from Cas12a orthologs. Data are shown as mean ± s.e.m. (n = 3); **P < 0.01, ***P < 0.001 (Mann-Whitney). e Efficiencies of targeted indel mutations introduced at five different human endogenous gene targets by CeCas12a and BfCas12a. Indel frequencies of PD1, HBB, IL12A, DNMT1, and TRAC were measured by T7E1 assay. f Summaries of the activities of CeCas12a at TTTV PAMs at a diverse panel of target sites in HEK293T cells. For indel percentages, each column represents the mean of n = 3 transfected cell cultures. Indel frequencies were analyzed by T7E1 assay. g Comparison of CeCas12a and Cas12a orthologs’ gene-editing efficiencies. Three target sites are from VEGFA loci. Indel frequencies analyzed by T7E1 assay. h Cas12a-mediated multiplex gene editing in human cells. Schematic of CRISPR array construct with the U6 direct repeat spacer cassette containing B2M, CTLA4, TRAC, and TRBC protospacer sequences (top). Indel frequencies were analyzed by T7E1 assay after transfection with CRISPR array and Cas12a plasmids (bottom)