Continuous evolution of SpCas9 variants compatible with non-G PAMs

Miller, Shannon M.; Wang, Tina; Randolph, Peyton B.; Arbab, Mandana; Shen, Max W.; Huang, Tony P.; Matuszek, Żaneta; Newby, Gregory A.; Rees, Holly A.; Liu, David R.

doi:10.1038/s41587-020-0412-8

Cited by 268 publications

(200 citation statements)

References 71 publications

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“…Our data in primary adult stem cells corroborate findings in cancer cell lines and mouse cortical neurons, in which prime editing typically offers greater precision than Cas9-mediated HDR and lower on-target efficiency than base editing, when the target nucleotide is suitably located 4 . Base editing, however, has been subject to various stages of optimization, whereas prime editing is still in its infancy 13 , 18 , 19 . The efficiency of prime editing in mammalian and plant cells has been strongly associated with pegRNA design, but optimal PBS and RT-template parameters remain elusive and differ between target sites 4 , 6 , 7 .…”

Section: Discussionmentioning

confidence: 99%

Prime editing for functional repair in patient-derived disease models

et al. 2020

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Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β‐catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.

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Section: Discussionmentioning

confidence: 99%

Prime editing for functional repair in patient-derived disease models

et al. 2020

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“…Our data in primary adult stem cells corroborate findings in cancer cell lines and mouse cortical neurons, in which prime editing typically offers greater precision than Cas9-mediated HDR and lower on-target efficiency than base editing, when the target nucleotide is suitably located 4 . Base editing, however, has been subject to various stages of optimization, whereas prime editing is still in its infancy 13,18,19 . The efficiency of prime editing in mammalian and plant cells has been strongly associated with pegRNA design, but optimal PBS and RT-template parameters remain elusive and differ between target sites 4,6,7 .…”

Section: Discussionmentioning

confidence: 99%

Prime editing for functional repair in patient-derived disease models

Schene

Joore

Oka

et al. 2020

Preprint

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15Prime editing is a novel genome editing technology using fusion proteins of Cas9-nickase 16 and reverse transcriptase, that holds promise to correct a wide variety of genetic defects. 17We succeeded in efficient prime editing and functional recovery of disease-causing 18 mutations in patient-derived liver and intestinal stem cell organoids. 19 20 Main 21The development of gene-editing therapies to treat monogenic diseases has long been an essential 22 goal of CRISPR/Cas9 research. Cas9-mediated homology-directed repair (HDR) can create all desired 23 base substitutions, insertions and deletions (indels). However, HDR relies on introduction of double-24 stranded DNA breaks, is inefficient and error-prone 1,2 . Base editing, that uses Cas9-nickases fused to 25 DNA-modifying enzymes, is more efficient and accurate than HDR, but can only correct four out of 26 twelve point mutations and no small insertions and deletions. Furthermore, base editing requires a 27 suitable protospacer adjacent motif (PAM) and the absence of co-editable nucleotides 3 . 28Prime editing combines a nicking-Cas9-reverse transcriptase fusion protein (PE2) with a prime 29 editing guide RNA (pegRNA) containing the desired edit. The pegRNA-spacer guides the formation of 30 a nick in the targeted DNA strand. The pegRNA-extension binds to this nicked strand and instructs 31 the synthesis of an edited DNA flap. This edited flap is then integrated by DNA repair mechanisms, 32 which can be enhanced by simultaneous nicking of the non-edited strand (Supplementary Fig. 1) 4 . 33Prime editing has been applied in human cancer cell lines and plant cells, but not in human disease 34 models 4,5,6 . Adult stem cell-derived organoids exhibit important functional properties of organs, 35allowing modeling of monogenic diseases 7 . We set out to develop and test prime editing in primary 36 patient-derived organoids. 38We first optimized prime editing for organoid cells using deletions and single-nucleotide 39 substitutions previously performed in HEK293T cells 4 . The non-edited strand was nicked by a second 40 'nicking sgRNA' to enhance editing (PE3). Our optimized protocol consisted of co-transfection of 41 prime edit plasmids with a GFP-reporter plasmid allowing selection and subsequent clonal expansion 42 of transfected cells. Prime editing of intestinal and ductal liver organoids resulted in efficient (>50%) 43 deletion of 5 nucleotides in HEK3 ( Fig. 1b and Supplementary Fig. 2). Furthermore, we were able to 44 induce a transversion mutation located 26 nucleotides downstream of the nick with substantial 45 efficiency (20%) (Fig. 1c). These results show successful prime editing of primary stem cells with 46 similar efficiency as observed in human cancer cell lines.Next, we targeted the Wnt-pathway intermediate ß-catenin (CTNNB1) in organoids. Activating 48 carcinogenic CTNNB1 mutations are found in ±40% of hepatocellular carcinoma, resulting in Wnt-49 signaling independent of exogenous stimuli 8 . We designed PE3 plasmids, containing pegRNA-50 extensions wi...

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“…Recent advances in PACE have introduced a split intein strategy, where one has the ability to encode an omega subunit on a complementary plasmid that is akin to the AP, such that the omega subunit would no longer be subject to selective pressure. 33 , 34 Excision of the omega subunit and subsequent addition to the protein of interest can be mediated by the addition of small molecules to create the functional translational unit that can then mediate gIII expression (please refer to ref ( 33 ) for a thorough description of this setup, as it is beyond the scope of this paper).…”

Section: Resultsmentioning

confidence: 99%

Phage-Assisted Continuous Evolution (PACE): A Guide Focused on Evolving Protein–DNA Interactions

et al. 2020

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The uptake of directed evolution methods is increasing, as these powerful systems can be utilized to develop new biomolecules with altered/novel activities, for example, proteins with new catalytic functions or substrate specificities and nucleic acids that recognize an intended target. Especially useful are systems that incorporate continuous evolution, where the protein under selective pressure undergoes continuous mutagenesis with little-to-no input from the researcher once the system is started. However, continuous evolution methods can be challenging to implement and a daunting investment of time and resources. Our intent is to provide basic information and helpful suggestions that we have gained from our experience with bacterial p hage- a ssisted c ontinuous e volution (PACE) toward the evolution of proteins that bind to a specific DNA target. We discuss factors to consider before adopting PACE for a given evolution scheme with focus on the PACE bacterial one-hybrid selection system and what optimization of a PACE selection circuit may look like using the evolution of the DNA-binding protein ME47 as a case study. We outline different types of selection circuits and techniques that may be added onto a basic PACE setup. With this information, researchers will be better equipped to determine whether PACE is a valid strategy to adopt for their research program and how to set up a valid selection circuit.

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Continuous evolution of SpCas9 variants compatible with non-G PAMs

Cited by 268 publications

References 71 publications

Prime editing for functional repair in patient-derived disease models

Prime editing for functional repair in patient-derived disease models

Prime editing for functional repair in patient-derived disease models

Phage-Assisted Continuous Evolution (PACE): A Guide Focused on Evolving Protein–DNA Interactions

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