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. 2021 Sep 30;11:19482. doi: 10.1038/s41598-021-98965-y

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© The Author(s) 2021

Open Access This 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/.

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Cas12a HDR gRNA selection and strand preference. (A) Schematic representation of targeting (T) and non-targeting (NT) donor template designs. The T strand is complementary to the gRNA sequence, whereas the NT strand contains the guide and PAM sequence. (B) HDR donors were designed with an EcoRI insert sequence positioned at varying distances from the from the first base of the Cas12a guide RNA ranging from 10 bases in the 5’ direction to 45 bases in the 3' direction for five genomic loci and delivered to HEK293 cells. RNP complexes (Alt-R A.s. Cas12a nuclease complexed with Alt-R CRISPR–Cas12a crRNA) were delivered at 5 µM along with 3 µM Alt-R Cpf1 Electroporation Enhancer and 3 µM donor template by nucleofection. HDR rates were assessed via EcoRI cleavage of targeted amplicons. The 21-base region where the gRNA targets is highlighted in green. The 4 base ‘TTTV’ PAM is highlighted in red. The gray shading indicates the confidence of fit. (C) An EcoRI restriction digest recognition site was inserted at position 16 of the gRNA sequence in 15 genomic loci in Jurkat and HAP1 cells using either the T or NT strand as the donor template and the combined results are graphed together. RNP complexes (Alt-R A.s. Cas12a Ultra nuclease complexed with Alt-R CRISPR–Cas12a crRNA) were delivered at 1 µM along with 3 µM Alt-R Cpf1 Electroporation Enhancer and 3 µM donor template by nucleofection. Total editing and perfect HDR was assessed via NGS. (D) Donors for two genomic loci were designed to insert an EcoRI site within the Cas12a guide sequence (position 15 of the guide) or outside of the guide sequence (24 bases from the start of the guide). ssODNs for these two insert locations were designed with blocking mutations in the PAM or guide sequence. The positions where blocking mutations were incorporated are indicated. (E) Donor templates for two genomic loci in HPRT1 were tested in Jurkat and Hela cells. RNP complexes (Alt-R A.s. Cas12a Ultra complexed with Alt-R CRISPR–Cas12a crRNA) were delivered at 2 µM along with 2 µM Alt-R Cpf1 Electroporation Enhancer and 3 µM donor template by nucleofection. HDR rates were assessed via NGS. Perfect HDR (blue), imperfect HDR (red) and total editing, which includes NHEJ events (black) are shown. Data are represented as mean ± SEM.