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Abstract

The use of single-stranded oligodeoxynucleotide (ssODN) along with CRISPR–Cas9-mediated double-strand breaks (DSB) is one of the most commonly deployed methods for introducing genetic alterations, but this approach has notable limitations. Recognizing this, we have developed a protocol article that provides a step-by-step process of donorguide, a covalent fusion of trans-activating CRISPR RNA (tracrRNA) and ssODN. Donorguide has the potential to enhance the introduction of specific genetic alterations (insertion, deletion, and substitution) at a DSB, improving homology-directed repair methods from zebrafish in vivo to human cells in vitro. We also explored and discuss the impact of increasing the length of donorguide homology arms in zebrafish.

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References

Chu

, Weber

, Wefers

, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol, 2015; 33(5):543–548; doi: 10.1038/nbt.3198

Maruyama

, Dougan

, Truttmann

, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol, 2015; 33(5):538–542; doi: 10.1038/nbt.3190

Miyaoka

, Berman

, Cooper

, et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep, 2016; 6:23549; doi: 10.1038/srep23549

Auer

, Duroure

, De Cian

, et al. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res, 2014; 24(1):142–153; doi: 10.1101/gr.161638.113

Shen

, Loh

, Shen

, et al. CRISPR as a strong gene editing tool. BMB Rep, 2017; 50(1):20–24; doi: 10.5483/bmbrep.2017.50.1.128

Simone

, Lee

, Daby

, et al. Chimeric RNA: DNA TracrRNA improves homology-directed repair in vitro and in vivo. Crispr J, 2022; 5(1):40–52; doi: 10.1089/crispr.2021.0087

Liu

, Zhang

, Xin

, et al. Global detection of DNA repair outcomes induced by CRISPR-Cas9. Nucleic Acids Res, 2021; 49(15):8732–8742; doi: 10.1093/nar/gkab686

Hendriks

, Clevers

, Artegiani

. CRISPR-Cas tools and their application in genetic engineering of human stem cells and organoids. Cell Stem Cell, 2020; 27(5):705–731; doi: 10.1016/j.stem.2020.10.014

Bertoni

. Methods for the assessment of ssODN-mediated gene correction frequencies in muscle cells. Methods Mol Biol, 2014; 1114:59–83; doi: 10.1007/978-1-62703-761-7_5

10.

Komor

, Kim

, Packer

, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016; 533(7603):420–424; doi: 10.1038/nature17946

11.

Anzalone

, Randolph

, Davis

, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 2019; 576(7785):149–157; doi: 10.1038/s41586-019-1711-4

12.

Jeong

, Song

, Bae

. Current status and challenges of DNA base editing tools. Mol Ther, 2020; 28(9):1938–1952; doi: 10.1016/j.ymthe.2020.07.021

13.

Gaudelli

, Komor

, Rees

, et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature, 2017; 551(7681):464–471; doi: 10.1038/nature24644

14.

Newby

, Liu

. In vivo somatic cell base editing and prime editing. Mol Ther, 2021; 29(11):3107–3124; doi: 10.1016/j.ymthe.2021.09.002

15.

Huang

, Newby

, Liu

. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat Protoc, 2021; 16(2):1089–1128; doi: 10.1038/s41596-020-00450-9

16.

Zhou

, Su

, Long

, et al. A universal strategy for AAV delivery of base editors to correct genetic point mutations in neonatal PKU mice. Mol Ther Methods Clin Dev, 2022; 24:230–240; doi: 10.1016/j.omtm.2022.01.001

17.

Jang

, Jo

, Lee

, et al. High-purity production and precise editing of DNA base editing ribonucleoproteins. Sci Adv, 2021; 7(35); doi: 10.1126/sciadv.abg2661

18.

Rees

, Komor

, Yeh

, et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun, 2017; 8:15790; doi: 10.1038/ncomms15790

19.

Vakulskas

, Dever

, Rettig

, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med, 2018; 24(8):1216–1224; doi: 10.1038/s41591-018-0137-0

20.

Kim

, Kim

, Cho

, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res, 2014; 24(6):1012–1019; doi: 10.1101/gr.171322.113

21.

Charlesworth

, Deshpande

, Dever

, et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med, 2019; 25(2):249–254; doi: 10.1038/s41591-018-0326-x

22.

Mehta

, Merkel

. Immunogenicity of Cas9 Protein. J Pharm Sci, 2020; 109(1):62–67; doi: 10.1016/j.xphs.2019.10.003

23.

Gammage

, Viscomi

, Simard

M-L

, et al. Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo. Nat Med, 2018; 24(11):1691–1695; doi: 10.1038/s41591-018-0165-9

24.

Russell

, Walton

KAC

, Whittaker

, et al. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science, 2020; 368(6488):290–296.

25.

Christie

, Kleinstiver

. Making the cut with PAMless CRISPR-Cas enzymes. Trends Genet, 2021; 37(12):1053–1055; doi: 10.1016/j.tig.2021.09.002

26.

Ghosh

, Myacheva

, Riester

, et al. Chimeric oligonucleotides combining guide RNA and single-stranded DNA repair template effectively induce precision gene editing. RNA Biol, 2022; 19(1):588–593; doi: 10.1080/15476286.2022.2067713

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Refined Homology-Directed Repair Methodological Approaches of Donorguide,a Chimeric RNA: DNA tracrRNA

Abstract

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References

Supplementary Material