Abstract
Precise and accurate gene correction is crucial for enabling iPSC-based therapies, and Cas9-Nickase based approaches are increasingly considered for in vivo correction of diseases such as beta-thalassemia. Here, we generate footprint-free induced pluripotent stem cells from a patient with a beta-thalassemia mutation (IVSII-1 G > A) and employ a double Cas9nickase-mediated correction strategy combined with a piggyBac transposon-modified donor vector for gene correction. Our approach further aimed to minimize the formation of adjacent single-strand breaks at the targeted allele through the destruction of the binding site for one guide and the use of a synonymous protospacer adjacent motif blocking mutation (canonical PAM sequence 5'-NGG-3' is changed to 5'-NCG-3', where N indicates any nucleobase) for the other guide. We show that this strategy indeed not only permits bi-allelic seamless repair of the beta-globin gene splice site mutation and negligible off-target mutagenesis or re-editing of the targeted allele but also results in unexpected on-target mutagenesis with some guide RNAs (gRNAs) in several targeted clones. This study thus not only validates a framework for seamless gene correction with enhanced specificity and accuracy but also highlights potential safety concerns associated with Cas9-nickase based gene correction.
Highlights
Et al, 2014; Song et al, 2015; Xu et al, 2015; Niu et al, 2016; Yang et al, 2016; and Wattanapanitch et al, 2018)
We show that this strategy permits bi-allelic seamless repair of the beta-globin gene splice site mutation and negligible off-target mutagenesis or re-editing of the targeted allele and results in unexpected ontarget mutagenesis with some guide RNAs in several targeted clones
The PAM sequence was inactivated at the targeted allele in the first pair, while the binding site for the first guide in the second pair was destroyed following the HDR event by the placement of the selection cassette. (c) Schematic of the location of the SNPs introduced in the donor template which include (1) a PAM blocking mutation to prevent CRISPR-Cas9n from generating a closely spaced single strand breaks (SSBs) in the donor template or the corrected allele when sgRNA1/3 is used (SNP1, green arrow); (2) a tracer SNP to validate that the modification occurred through recombination (SNP2, green arrow); and (3) a base substitution that corrects the disease-causing mutation
Summary
Et al, 2014; Song et al, 2015; Xu et al, 2015; Niu et al, 2016; Yang et al, 2016; and Wattanapanitch et al, 2018). HDR accuracy can theoretically be further improved by introducing a single nucleotide polymorphisms (SNP) in the protospacer adjacent motif (PAM) sequence at the binding site for one of the guides of the pair set, as shown by Paquet et al (Paquet et al, 2016) for Cas9WT The effectiveness of both strategies relies on the high-fidelity repair of the single-strand breaks (SSBs). We combine all of the above approaches to correct a HBB gene splice mutation in beta-thalassemia iPSCs, a locus that appears susceptible to off-target activity for some sgRNA sequences (Cradick et al, 2013 and Xu et al, 2015) We demonstrate that this combined strategy can prevent undesired ontarget indels for some guides and reveal an unexpectedly high rate of mutagenesis associated with the stand-alone action of Cas9n at one of the sgRNA sites, as indicated by the presence of an indel at the binding site of one of the guides. This highlights that despite inbuilt safety features, CRISPR Cas9-approaches need to be carefully evaluated for each gene and guide before progressing to in vivo gene correction in patients
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