Genomics and Applied Biology 2024, Vol.15, No.2, 99-106 http://bioscipublisher.com/index.php/gab 101 Figure 1 CRISPR/Cas9 editing confirmation and efficiency comparisons of different U6 promoters by analysis of NANOS3 gRNA target (Adopted from Hamar and Kültz, 2020) Image caption: (aand b) Sequencing results of individual alleles from plasmid sub-cloned test amplicons. (a) For the NANOS3 target, 17 out of 21 amplicons (81%) were altered at the target site (4 wild-type sequences not shown). (b) For the NFAT5 T10 target, 3 out of 19 amplicons (16%) were altered at the target site. The remaining 16 sequences were identical to the O. niloticus reference sequence except for an extra nucleotide (an A when reading the gene sequence 5′ to 3′, a T when reading the gRNA sequence 5′ to 3′) 5 base pairs from the PAM sequence (highlighted in green). (c) Alternate cloning strategy for changing gRNA target sequence in expression vector illustrated. Utilizes a mutated TU6 (TU6m) in which a single nucleotide was changed adjacent to the TSS generating a ClaI restriction site. The TU6m is included in the base vector in which new gRNA target sequences can be added by annealed oligos. (d) Mutational efficiency quantified by TIDE indel% analysis of four different U6 promoters using the same gRNA target (NANOS3) showing superior editing obtained from both versions of the tilapia U6 promoters (over fivefold over all others). The Human and Zebrafish U6 promoters were not statistically significant from the no U6 control. Gene maps and DNA sequence images were generated using Geneious 11.0.3 (Biomatters, https://www.geneious.com). Bar plot was generated using Rstudio version 1.1.456 (https://rstudio.com). Image editing and assembly into complete figures was performed using Inkscape version 0.92 (https://www.inkscape.org) (Adopted from Hamar and Kültz, 2020)
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