A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines

Eggenschwiler, Reto; Gschwendtberger, Thomas; Felski, Christian; Jahn, Christopher; Sterneckert, Jared; Hermann, Andreas; Lühmann, Jonathan Lukas; Steinemann, Doris; Haase, Alexandra; Petri, Susanne; Cantz, Tobias

doi:10.1038/s41598-021-01689-2

Cited by 24 publications

(22 citation statements)

References 25 publications

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“…Some prime editing applications, such as pooled screening, require stable cellular expression of PE from a genomically integrated cassette. PiggyBac transposase and lentiviruses can stochastically insert DNA encoding PEs and pegRNAs into the genome and mediate high editing efficiencies in human cell lines, induced pluripotent stem cells (iPSCs) and mouse cortical neurons 52,55,[109][110][111] . Similarly, human iPSC lines have been generated with inducible PE2 at the AAVS1 safe harbour locus by HDR-mediated integration 82,112,113 .…”

Section: Dna Transfection and Viral Deliverymentioning

confidence: 99%

Prime editing for precise and highly versatile genome manipulation

2022

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single-stranded DNA (ssDNA) at the target site, triggering deamination by the tethered ssDNA-specific deaminase enzyme. Cytosine base editors (CBEs) and adenine base editors (ABEs) contain deaminases that catalyse C•G-to-T•A and A•T-to-G•C changes, respectively [27][28][29][30][31] . C•G-to-G•C base editors (CGBEs) are similar to CBEs, but stimulate replacement of the deaminated cytosine base with guanine, albeit with lower typical efficiencies and product purities compared to CBEs and ABEs [33][34][35][36] . Compared to Cas nucleases, base editors exhibit substantially greater efficiency with few indel byproducts, and show far fewer undesired consequences of DSBs than nucleases in side-by-side comparisons 19,21,[23][24][25][26]37,38 .Because base editors deaminate within a small window of 4-5 nucleotides (nt) canonically, C or A nucleotides very close to the target C or A can also undergo conversion, resulting in 'bystander editing'. The application of base editors to make changes in the coding sequence of genes usually results in only synonymous mutations within a typical base editing activity window 39 owing to the frequency of transition mutations being silent in the genetic code. Nevertheless, undesired base editing of bystander nucleotides can be challenging to avoid in some cases. Base-editing activity is also restricted by the targeting scope of the Cas domain, which requires the presence of a protospacer adjacent motif (PAM) sequence at a specific distance range (typically 15 ± 2 nt) from the target nucleotide. Moreover, some base editors can induce off-target mutations in DNA and RNA [40][41][42] . Although engineering efforts have mitigated many of these drawbacks [43][44][45][46][47][48][49][50][51] , current base editors can only make six of the 12 possible types of point mutation, leaving many other classes of possible DNA edits, such as insertions, deletions and most transversions, beyond the reach of base editing.Motivated by the need for a precise and highly versatile geneediting technology, prime editing enables all types of DNA substitution, small insertions and small deletions to be installed at targeted sites in the genomes of living cells without directly forming DSBs 52 (Fig. 1c). Prime editing minimally requires a prime editor (PE) protein, which is typically a fusion of a nickase Cas9 (nCas9) and a reverse transcriptase (RT), along with a prime editing guide RNA (pegRNA), which specifies the target site for the edit and contains a programmable RNA template for the desired DNA sequence change. To mediate editing, PEs nick the target site on the genome and extend new DNA sequence from this nick using the pegRNA as a template (Fig. 2). This edited DNA strand is then incorporated into the genome through endogenous cellular processes that can be promoted by also nicking the non-edited strand 52 .Through this mechanism of directly rewriting target DNA, prime editing offers extraordinarily high versatility, editing purity and DNA target specificity in comparison to base editing and HDR with nucl...

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Section: Dna Transfection and Viral Deliverymentioning

confidence: 99%

Prime editing for precise and highly versatile genome manipulation

2022

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“…remain integrated in the genome; thus, this system is not scarless. Eggenschwiler et al presented a similar system, but with the ability to excise the prime editing components following the editing step using the piggyBac system for scarless editing [ 45 ].…”

Section: Strategies To Increase Hdr-dependent Crispr-cas9 Mediated Ge...mentioning

confidence: 99%

Selecting for CRISPR-Edited Knock-In Cells

Reuven

Shaul

2022

IJMS

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CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, “scarless” selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.

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“…For example, Bharucha et al [23] developed a doxycycline-inducible-PE2 expressing iPSC line that provides a fast and efficient approach. Similarly, the PE construct was integrated into the genome via a piggyBac transposon, enabling sustained expression of the protein and efficient edit in iPSC cells [24]. Finally, Habib et al [25] created a drug-inducible PE2-expressing human embryonic stem cell (hESC) line to introduce small indels and all base substitutions and showed that long-term overexpression of PE2 did not generate significant frequencies of genome-wide SNVs or indels.…”

Section: Advantages Of Prime Editingmentioning

confidence: 99%

The potential of CRISPR-Cas9 prime editing for cardiovascular disease research and therapy

Bharucha

Arias

Karakikes

2022

Current Opinion in Cardiology

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Purpose of reviewThe ability to edit any genomic sequence has led to a better understanding of gene function and holds promise for the development of therapies for genetic diseases. This review describes prime editing - the latest CRISPR-Cas9 genome editing technology. Prime editing enables precise and accurate genome editing in terminally differentiated, postmitotic cells like cardiomyocytes, paving the way for therapeutic applications for genetic cardiomyopathies.Recent findingsPrime editing has been used to precisely insert up to 40 bases, create deletions up to 80 base pairs, and can perform all 12 possible transition and transversion base mutations with lower indels and off-target effects than other genome editing methods. The development of several software tools has simplified the experimental design and led to increased efficiency of the process. Improvements in methods for in-vivo delivery of the prime editing components should enable this technology to be used to edit the genome in patients.SummaryPrime editing has the potential to revolutionize the future of biomedical research and transform cardiovascular medicine. Improved understanding of the prime editing process and developments in agent design, efficacy and delivery will benefit scientists and patients and could be an effective way to cure cardiovascular diseases.

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A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines

Cited by 24 publications

References 25 publications

Prime editing for precise and highly versatile genome manipulation

Prime editing for precise and highly versatile genome manipulation

Selecting for CRISPR-Edited Knock-In Cells

The potential of CRISPR-Cas9 prime editing for cardiovascular disease research and therapy

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