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Abstract: The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, spRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for spRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of spRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of spRY for human genome editing.

SpRY能识别人类细胞中前间区序列邻近基序吗？

[1]AsanoY, YamashitaK, HasegawaA, et al., 2021. Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum. Sci Rep, 11:11163.

[2]CrooksGE, HonG, ChandoniaJM, et al., 2004. WebLogo: a sequence logo generator. Genome Res, 14(6):1188-1190.

[3]EvansBA, BernsteinDA, 2021. SpRY Cas9 can utilize a variety of protospacer adjacent motif site sequences to edit the Candida albicans genome. mSphere, 6(3):e00303-21.

[4]FarehM, ZhaoW, HuWX, et al., 2021. Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance. Nat Commun, 12:4270.

[5]HeXB, WangYF, YangFY, et al., 2019. Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNA^Gln-processing system in human cells. J Biol Chem, 294(23):9308-9315.

[6]HiranoS, NishimasuH, IshitaniR, et al., 2016. Structural basis for the altered PAM specificities of engineered CRISPR-Cas9. Mol Cell, 61(6):886-894.

[7]HsuPD, ScottDA, WeinsteinJA, et al., 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31(9):827-832.

[8]HsuPD, LanderES, ZhangF, 2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6):1262-1278.

[9]HuJH, MillerSM, GeurtsMH, et al., 2018. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 556(7699):57-63.

[10]JiangFG, DoudnaJA, 2017. CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys, 46:505-529.

[11]JinekM, ChylinskiK, FonfaraI, et al., 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bac‍terial immunity. Science, 337(6096):816-821.

[12]KomorAC, BadranAH, LiuDR, 2017. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell, 168(1-2):20-36.

[13]LiJ, XuRF, QinRY, et al., 2021. Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants. Mol Plant, 14(2):352-360.

[14]MojicaFJM, Díez-VillaseñorC, García-MartínezJ, et al., 2009. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading), 155(Pt 3):733-740.

[15]NishimasuH, ShiX, IshiguroS, et al., 2018. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science, 361(6408):1259-1262.

[16]PinelloL, CanverMC, HobanMD, et al., 2016. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol, 34(7):695-697.

[17]ShahSA, ErdmannS, MojicaFJM, et al., 2013. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol, 10(5):891-899.

[18]SternbergSH, ReddingS, JinekM, et al., 2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507(7490):62-67.

[19]TangLC, YangFY, HeXX, et al., 2019. Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells. Cell Regen, 8(2):44-50.

[20]WaltonRT, ChristieKA, WhittakerMN, et al., 2020. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science, 368(6488):290-296.

[21]WaltonRT, HsuJY, JoungJK, et al., 2021. Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA. Nat Protoc, 16(3):1511-1547.

[22]XuZY, KuangYJ, RenB, et al., 2021. SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition. Genome Biol, 22:6.

[23]YangFY, LiuCB, ChenD, et al., 2017. CRISPR/Cas9-loxP-mediated gene editing as a novel site-specific genetic manipulation tool. Mol Ther Nucleic Acids, 7:378-386.

[24]ZhangWW, YinJH, Zhang-DingZR, et al., 2021. In-depth assessment of the PAM compatibility and editing activities of Cas9 variants. Nucleic Acids Res, 49(15):8785-8795.

[25]ZhangYL, GeXL, YangFY, et al., 2014. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci Rep, 4:5405.