Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors

Abstract

In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.

Development of CRISPR technology for precise single-base genome editing: a brief review

“…Primer editing of plant systems (PPE) was demonstrated in rice and wheat ( 46 ). The delivery of primer editing molecules in mRNA forms was also shown in human iPS cells ( 47 ) and in mouse embryos ( 48 ).…”

“…Therefore, application of base editors are unsuitable for mutagenesis of multiple bases within defined sequences that requires concomitant combinations of C-to-T and A-to-G conversions. To address the issue, an alternative DSB-free editing method, known as prime editing, was developed ( 21 , 46 - 48 ). Although prime editing is similar to base editing in that no DSBs are involved, a distinct molecular mechanism is involved ( Fig.…”

Prime Editing: Making the Move to Prime Time

“…1 The most significant advantage of PE over BE is the possibility of effective correction of not only point mutations but also indels, as well as combinations of these types of mutations (with efficiency up to 75%). [1][2][3][4][5][6][7][8][9] Unlike the classic CRISPR-Cas method, PE does not produce double-stranded DNA breaks (DSBs), creating only a nick (or two nicks in the case of PE3). In the absence of DSBs, the number of indels in the target DNA decreases in most cases to an undetectable level.…”

“…In the absence of DSBs, the number of indels in the target DNA decreases in most cases to an undetectable level. [1][2][3][4] PE3 forms two adjacent nicks that lead to the formation of a larger percentage of indels in the target locus than PE1 and PE2. Despite this, the non-homologous end joining/HDR ratio using CRISPR-Cas9 with ssODN as a donor molecule for HDR is 270 times higher than in PE3.…”

“…Over the past year, PE has been tested in various organisms and cell cultures: in humans [1][2][3][4] and mouse cells, 1,3 , mouse embryos, 3 and plants. [5][6][7][8][9] The editing efficiency (ranging from 0% to 75%) depends on several factors, such as the targeted gene, the lengths of the primer binding site (PBS) and RT template, the type of mutation, the localization of mutation relative to the protospacer adjacent motif (PAM), and the distance between nicks.…”