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CRISPR Prime Editing with Ribonucleoprotein Complexes in Zebrafish and Primary Human Cells

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CRISPR Prime Editing with Ribonucleoprotein Complexes in Zebrafish and Primary Human Cells BRIEF COMMUNICATION https://doi.org/10.1038/s41587-021-00901-y CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells Karl Petri 1,2,7, Weiting Zhang3,4,7, Junyan Ma3,4,5,7, Andrea Schmidts 4,6,7, Hyunho Lee1,2, Joy E. Horng 1,2, Daniel Y. Kim 1,2, Ibrahim C. Kurt 1,2, Kendell Clement 1,2, Jonathan Y. Hsu 1,2, Luca Pinello1,2, Marcela V. Maus 4,6, J. Keith Joung 1,2,8 ✉ and Jing-Ruey Joanna Yeh 3,4,8 ✉ Prime editors have been delivered using DNA or RNA vec- guide RNAs (Supplementary Note 2). RNP complexes were injected tors. Here we demonstrate prime editing with purified ribo- into single-cell zebrafish embryos incubated at 28.5 or 32 °C and nucleoprotein complexes. We introduced somatic mutations assessed for on-target prime editing 1 day following injection, using in zebrafish embryos with frequencies as high as 30% and targeted amplicon next-generation sequencing (NGS) (Fig. 1d). demonstrate germline transmission. We also observed unin- We observed three types of on-target edit: ‘pure prime edits’ tended insertions, deletions and prime editing guide RNA (PPEs)—alleles with only the intended edit, ‘impure prime edits’ (pegRNA) scaffold incorporations. In HEK293T and primary (IPEs)—alleles with both the intended edit and additional muta- human T cells, prime editing with purified ribonucleoprotein tions and ‘by-product edits’—alleles without the intended edit but complexes introduced desired edits with frequencies of up to with other mutations (Supplementary Note 3 and Supplementary 21 and 7.5%, respectively. Tables 1 and 2). The mean PPE and IPE frequencies were 0.53 and The recently described prime editing (PE) method can install all 0.45% for tyr_1, and 0.25 and 0.48% for tyr_2, respectively, though 12 nucleotide substitutions, short insertions and short deletions1,2. rates varied across the different conditions tested (Supplementary PE employs a pegRNA and a PE protein (PE2) consisting of a Cas9 Fig. 3a). In comparison, by-product frequencies were more uniform nickase (nCas9) fused to an engineered M-MLV reverse transcrip- across the different conditions (generally <10% for tyr_1 and <5% tase (RT) (Fig. 1a and Supplementary Note 1). PE has been used for tyr_2) (Supplementary Fig. 3a). IPE alleles had a wide range of to edit the genomes of mammalian cells and organoids in culture, unintended edits, including pegRNA scaffold incorporations, dele- plants, Drosophila and somatic cells in mice, with the PE compo- tions, potential RTT duplications/insertions and other insertions/ nents delivered as DNAs or RNAs1,3–7. Here we demonstrate that substitutions (Supplementary Note 3). Notably, deletions surround- purified PE ribonucleoprotein (RNP) complexes can induce somatic ing the 3′ boundary of the RT-synthesized DNA flap were common and germline-transmissible mutations in zebrafish and efficiently in IPE alleles (Fig. 1e and Supplementary Fig. 3b). By contrast, edit the genomes of human cell lines and human primary cells. by-product edits mostly harbored deletions centered around the We first constructed bacterial and mammalian expression plas- nicking site of PE2 (Fig. 1e and Supplementary Fig. 3b). The C9E mids encoding PE2 without or with a polyhistidine tag on its N- or pegRNA scaffold generally yielded higher PPE and IPE frequencies C-terminal ends (named PE2, His-PE2 and PE2-His, respectively), without substantial increase in by-product edits. Moreover, edit fre- and found that the untagged and tagged proteins elicited similar quencies of RTTs of 13–15 nt were comparable, or in some cases editing efficiencies or spectra of editing outcomes when expressed preferable, to longer lengths whereas the effects of PBS length and in human HEK293T cells (Supplementary Fig. 1a,b). Subsequently, temperature were more variable (Supplementary Note 2). we overexpressed and purified full-length PE2-His protein from Next, we compared the PE2 and PE3 strategies (Supplementary Escherichia coli (Fig. 1b) and verified that purified PE2-His, together Note 1) in zebrafish, with PBS lengths of 10 and 13 nt and incuba- with in vitro transcribed pegRNA, could direct site-specific DNA tion temperatures of 28.5 and 32 °C (Fig. 1f). The pegRNAs were nicks and carry out pegRNA-templated reverse transcription designed to introduce single G → C or G → T point mutations in vitro (Fig. 1c and Supplementary Fig. 2a–c). Amplification and into eight different zebrafish genes. All pegRNAs used the C9E sequencing of these reaction products showed that PE2 can reverse scaffold, had 14- or 15-nt RTT lengths and targeted sites that we transcribe the entire pegRNA, including pegRNA scaffold and had determined could be efficiently mutated with Cas9 protein spacer sequences (Supplementary Fig. 2c–e). (Supplementary Fig. 4). We observed PPEs and IPEs at variable fre- Next, we tested whether PE RNP complexes with PE2-His could quencies (Supplementary Fig. 5), and by-product edits at generally mediate targeted point mutations in zebrafish embryos at the higher frequencies than PPEs and IPEs across all conditions tested tyr_1 and tyr_2 sites in the zebrafish tyr gene using the PE3 strat- at the target sites (Supplementary Fig. 5). Comparative analyses egy (Supplementary Note 1). We used pegRNAs harboring primer of these data revealed that certain conditions modestly improved binding sites (PBSs) and RT templates (RTTs) of various length PPE frequencies without proportional increase in IPE frequencies and, for the tyr_1 pegRNAs, two different pegRNA architectures or appreciable impact on by-product edit frequencies: incubation (C9 (ref. 8) and C9E (ref. 9)), together with their matched nicking at 32.0 °C was better than at 28.5 °C, and pegRNA PBS length of 1Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA. 2Department of Pathology, Harvard Medical School, Boston, MA, USA. 3Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA. 4Department of Medicine, Harvard Medical School, Boston, MA, USA. 5Medical College, Dalian University, Dalian, China. 6Cellular Immunotherapy Program, Massachusetts General Hospital Cancer Center, Charlestown, MA, USA. 7These authors contributed equally: Karl Petri, Weiting Zhang, Junyan Ma, Andrea Schmidts. 8These authors jointly supervised this work: J. Keith Joung, Jing-Ruey Joanna Yeh. ✉e-mail: [email protected]; [email protected] NATURE BIOTechnOLOGY | www.nature.com/naturebiotechnology BRIEF COMMUNICATION NATURE BIOTECHNOLOGY 10 nt was better than 13 nt (Fig. 1g). Outcomes with the PE3 strategy transmitted the desired PPE to 8.3% of its offspring, and a second did not appear to be consistently better than with PE2 (Fig. 1g,h), founder that transmitted an IPE to 7.1% of its offspring (Fig. 2b,c contrary to that reported in human cell lines1. Varying the pegRNA/ and Supplementary Table 3). Similarly, for the kras G12V muta- ngRNA ratio or deploying the PE3b strategy (Supplementary tion, we screened 14 adult F0 fish and identified one founder that Note 1) also did not consistently improve PPE frequencies relative transmitted the desired PPE to 12.3% of its offspring and a second to PE2 (Supplementary Fig. 6a–c and Supplementary Note 4). We founder that transmitted an IPE to 7.1% of its offspring (Fig. 2b,c and speculate that the effects of PE3 may depend on cell division rate Supplementary Table 4). Interestingly, both germline-transmitted and cellular DNA repair machinery differences between zebraf- IPE alleles harbored a single C-to-G or A-to-G substitution at the ish embryos and human cells (Supplementary Note 4). Overall, first base 3′ to the RTT-encoded sequence, perhaps reflecting prov- pegRNAs with PBS length of 10 nt and embryos incubated at 32.0 °C enance from pegRNA scaffold incorporations (Supplementary Note generally yielded higher PPE frequencies (mean ranges of 0.46–4.01 3). Efficient germline transmission of both pathogenic variants that and 0.28–2.06% with PE2 and PE3, respectively; Fig. 1h), without we attempted to install demonstrates that PE can be used to create proportional increases in undesired IPE or by-product frequencies. zebrafish models of human disease. To investigate the efficiency of PE2 for creation of short, Because gene-editing nuclease-induced homology-directed defined-length deletions and insertions in zebrafish, we used repair (HDR) can also be used to create the same modifications that pegRNAs designed to introduce 5-base pair (bp) or 10-bp deletions we installed using PE, we compared the efficiencies and precision of at three sites and insertions of 3–18 bp at two sites. We obtained PE and HDR in the introduction of targeted base substitutions, inser- mean PPE frequencies ranging from 4.13 to 33.61% for precise tions, and deletions at two sites. The results show that PE induced deletions (Fig. 1i), and from 0.10 to 18.00% for precise insertions all three types of intended mutation at frequencies comparable to or (Fig. 1j), which were higher than PPE frequencies for point muta- higher than Cas9-mediated HDR (Supplementary Fig. 10). PE con- tions at the same target sites (Fig. 1h). Because the intended edits in sistently induced fewer unwanted edits than Cas9-mediated HDR these experiments were deletions or insertions, we could not always (Supplementary Fig. 10), demonstrating the relatively higher preci- distinguish between IPEs and the insertions and deletions (indels) sion of the PE method. included in by-product edits, but the frequencies of all of these To determine whether PE could induce unwanted off-target ‘non-PPE’ alleles were comparable to or higher than those of the mutations in zebrafish, we used targeted amplicon NGS to assess PPEs (Supplementary Figs. 7a and 8a,b). The majority of non-PPE all sites in the zebrafish reference genome (GRCz11) with three or alleles are larger deletions or deletions close to the pegRNA nicking fewer mismatches relative to the on-target sites of five pegRNAs site or the 3′ end of the DNA sequence complementary to the RTT (Methods; Supplementary Table 5).
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