Prime editing primarily induces undesired outcomes in mice

Aida, Tomomi; Wilde, Jonathan J.; Yang, Li; Hou, Yong; M, Li; D, Xu; J, Lin; Qi, Peng; Zhang, Lu; Feng, Guoping

doi:10.1101/2020.08.06.239723

Cited by 41 publications

(49 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Prime editing in plant protoplasts also produces a fraction of undesired editing outcomes when employing either the PE2 and PE3 strategy 7 . Interestingly, in mouse zygotes the PE3 strategy produces alleles containing the desired edit, but a large fraction also harbor deletions of various sizes between the target site and the nicking site 11 . Reassuringly, for our in vivo PE3 editing the majority of modified alleles contain the intended product without additional modifications.…”

Section: Discussionmentioning

confidence: 99%

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

Liu

Liang

Zheng

et al. 2020

Preprint

View full text Add to dashboard Cite

Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.

show abstract

Section: Discussionmentioning

confidence: 99%

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

Liu

Liang

Zheng

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…It would also be of interest to evaluate whether other genome editing strategies, such as prime and base editing, nickases or improvements in the efficiency of integrating a repair template, may reduce the on-target complexities observed by us and others using spCas9. However, non-negligible frequencies of editing-associated large deletions have been reported after the use of the Cas9 D10A nickase in mESCs (14) and prime editing in early mouse embryos (43). By contrast, while proof-of-principle studies suggest that base editors could be used to repair disease-associated mutations in human embryos, further refinements to reduce the likelihood of unexpected conversion patterns and high rates of off-target edits would be of benefit (2).…”

Section: Discussionmentioning

confidence: 99%

Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos

Alanis-Lobato

Zohren

McCarthy

et al. 2020

Preprint

View full text Add to dashboard Cite

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germline. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and Cas9-only control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss-of-heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in approximately 22% of the human embryo cells analysed and spanned 4 to 20kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

show abstract

“…Thus, in principle, prime editing represents a versatile, precision-guided platform that can potentially correct all SNVs of clinical importance [24]. Prime editing has been reported in plants [25][26][27], in early-staged mouse embryos [28,29], and in Drosophila [30]. However, there has yet to be a comparative analysis of prime editing versus CRISPR-mediated HDR editing in mice bred through the germline and analyzed for phenotypes.…”

Section: Introductionmentioning

confidence: 99%

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

et al. 2021

View full text Add to dashboard Cite

Background Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. Results Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. Conclusions PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

show abstract

Prime editing primarily induces undesired outcomes in mice

Cited by 41 publications

References 40 publications

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

Contact Info

Product

Resources

About