Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.
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This work was supported by the Hassenfeld Scholar Award (to J.-R.J.Y.), NIH no. R01 GM134069 (to J.-R.J.Y.), NIH no. RM1 HG009490 (to J.K.J. and L.P.), NIH no. R35 GM118158 (to J.K.J.), the Defense Advanced Research Projects Agency Safe Genes Program grant no. HR0011-17-2-0042 (to J.K.J.) and National Human Genome Research Institute Genomic Innovator Award no. R35 HG010717 (to L.P.). K.P. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 417577129. J.M. received support from the China Scholarship Council (no. 201808210354). A.S. received support from a John Hansen Research Grant from DKMS (no. DKMS-SLS-JHRG-2020-04). We thank K. K. Lam for technical assistance and K. K. Lam and J. Grünewald for discussions and technical advice. We thank L. Paul-Pottenplackel for help with revision of the manuscript.
J.K.J. has financial interests in Beam Therapeutics, Chroma Medicine (formerly known as YKY, Inc.), Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, SeQure Dx, Transposagen Biopharmaceuticals and Verve Therapeutics (formerly known as Endcadia). K.P. has a financial interests in SeQure Dx, Inc.. L.P. has financial interests in Edilytics, SeQure Dx, Inc. and Excelsior Genomics. K.P. and D.Y.K. are paid consultants to Verve Therapeutics. K.C. is an employee, shareholder and officer of Edilytics, Inc. The interests of J.K.J., L.P., K.C., K.P. and D.Y.K. were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. M.V.M. is an inventor on patents related to adoptive cell therapies, held by Massachusetts General Hospital and University of Pennsylvania (some licensed to Novartis). M.V.M. holds equity in TCR2 and Century Therapeutics, and has served as a consultant for multiple companies involved in cell therapies. The remaining authors declare no competing interests.
Peer review information Nature Biotechnology thanks Jia Chen, Ayal Hendel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Petri, K., Zhang, W., Ma, J. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00901-y