CRISPR–Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9–sgRNA complex as a guide, the majority of the 3′ end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3′-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA–RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.
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We thank T. Jacks, P. Sharp, Z. Weng, C. Mello, and E. Sontheimer for discussions, and Y. Li for technical assistance. We thank K. Joung (Massachusetts General Hospital and Harvard Medical School) for sharing U2OS-GFP-PEST cells and J. Smith and A. Sheel for proofreading. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512, DP2HL137167 and P01HL131471 (to W.X.). H.Y. is supported by 5-U54-CA151884-04 (NIH) and Skoltech Center. W.X. was supported by the Lung Cancer Research Foundation, Hyundai Hope on Wheels, and ALS Association. This work is supported in part by Cancer Center Support (core) grant P30-CA14051 from the NIH. We thank the Swanson Biotechnology Center at MIT for technical support.
H.Y., C.-Q.S., W.X., D.G.A and R.L. have applied for patents related to this study. D.G.A. is a scientific co-founder of CRISPR Therapeutics.
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Yin, H., Song, CQ., Suresh, S. et al. Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. Nat Chem Biol 14, 311–316 (2018). https://doi.org/10.1038/nchembio.2559
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