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.

In this work, we used a comprehensive set of the fusion oncogene RUNX1-RUNX1T1 alternative exons to analyze the patterns of its mRNA generation. We found that the waste majority of alternative exons are modified variants of canonical exons, and the transcripts, including such exons, have a very low expression level. The «hot regions», including exons 4a, 6, 8b, 9, 11 and 12, produces about 80 % of such variants. Also we described a new transcription start region of RUNX1-RUNX1T1 and provide the evidences of co-expression of the fusion RNAs with normal and shortened 3′-UTRs in leukemic cells.

Ultraconserved elements (UCEs) are genomic regions that are highly conserved among vertebrates and are under extreme purifying selection. Despite this extreme conservation, the biological importance of UCEs has remained elusive thus far. Here, we investigate the relevance of 93 UCEs for survival and fitness of mouse embryonic stem cells (mESCs) utilizing CRISPR/Cas9 technology. Surprisingly, systematic analyses of deletion clones revealed no measurable effects on mESC viability, proliferation or morphology.Furthermore, growth of homozygous UCE knockout clones under stress conditions or growth in direct competition with wild-type mESC revealed no phenotypic difference. Likewise, no difference in differentiation potential and on the expression level of key marker genes of differentiation was observed upon embryonic body formation. While functional overlap (redundancy) between several UCEs has been proposed to account for the absence of effects in single UCE knockouts, we obtained phenotypically normal mESC clones carrying up to five homozygous UCEs deletions, challenging this model. Finally, integration of additional UCE copies into the genome of mESCs also revealed no adverse effects. In summary, under all tested experimental conditions, UCEs appear to be dispensable for fitness, viability and differentiation potential of mESCs, despite their strong conservation.