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Volume 52 Issue 2
Feb.  2025
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Article Navigation >  Journal of Genetics and Genomics  > 2025  >  52(2): 258-268

PAMPHLET: PAM Prediction HomoLogous-Enhancement Toolkit for precise PAM prediction in CRISPR-Cas systems

doi: 10.1016/j.jgg.2024.10.014
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    a. Guangdong Provincial Key Laboratory of Interdisciplinary Research and Application for Data Science, BNU-HKBU United International College, Zhuhai, Guangdong 519087, China;

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    b. School of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong 510006, China;

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    c. BGI Research, Hangzhou, Zhejiang 310030, China;

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    d. BGI Research, Shenzhen, Guangdong 518083, China;

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    e. College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China;

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    f. Laboratory of Integrative Biomedicine, Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark;

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    g. Qingdao-Europe Advanced Institute for Life Sciences, BGI Research, Qingdao, Shandong 266555, China;

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    h. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, Zhejiang 310024, China;

  •  

    i. STOmics Americas Ltd., 2904 Orchard Pkwy, San Jose CA 95134, USA

  • Received Date: 2024-07-07
  • Accepted Date: 2024-10-26
  • Rev Recd Date: 2024-10-24
    • Available Online: 2025-07-11
  • Publish Date: 2024-11-08
    Abstract
  • CRISPR-Cas technology has revolutionized our ability to understand and engineer organisms, evolving from a singular Cas9 model to a diverse CRISPR toolbox. A critical bottleneck in developing new Cas proteins is identifying protospacer adjacent motif (PAM) sequences. Due to the limitations of experimental methods, bioinformatics approaches have become essential. However, existing PAM prediction programs are limited by the small number of spacers in CRISPR-Cas systems, resulting in low accuracy. To address this, we develop PAMPHLET, a pipeline that uses homology searches to identify additional spacers, significantly increasing the number of spacers up to 18-fold. PAMPHLET is validated on 20 CRISPR-Cas systems and successfully predicts PAM sequences for 18 protospacers. These predictions are further validated using the DocMF platform, which characterizes protein–DNA recognition patterns via next-generation sequencing. The high consistency between PAMPHLET predictions and DocMF results for Cas proteins demonstrates the potential of PAMPHLET to enhance PAM sequence prediction accuracy, expedite the discovery process, and accelerate the development of CRISPR tools.
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  • Abdelrahman, M., Wei, Z., Rohila, J.S.,Zhao, K., 2021. Multiplex genome-editing technologies for revolutionizing plant biology and crop improvement. Front. Plant Sci. 12, 721203.
    Armario Najera, V., Twyman, R.M., Christou, P.,Zhu, C., 2019. Applications of multiplex genome editing in higher plants. Curr. Opin. Biotechnol. 59, 93-102.
    Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A.,Horvath, P., 2007. Crispr provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712.
    Biswas, A., Gagnon, J.N., Brouns, S.J.J., Fineran, P.C.,Brown, C.M., 2013. Crisprtarget: bioinformatic prediction and analysis of crrna targets. RNA Biol. 10, 817-827.
    Bland, C., Ramsey, T.L., Sabree, F., Lowe, M., Brown, K., Kyrpides, N.C.,Hugenholtz, P., 2007. Crispr recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinf. 8, 209.
    Boudry, P., Semenova, E., Monot, M., Datsenko, K.A., Lopatina, A., Sekulovic, O., Ospina-Bedoya, M., Fortier, L.-C., Severinov, K., Dupuy, B., et al., 2015. Function of the crispr-cas system of the human pathogen clostridium difficile. mBio 6, e01112-e01115.
    Briner, A.E., Donohoue, P.D., Gomaa, A.A., Selle, K., Slorach, E.M., Nye, C.H., Haurwitz, R.E., Beisel, C.L., May, A.P.,Barrangou, R., 2014. Guide rna functional modules direct cas9 activity and orthogonality. Mol. Cell 56, 333-339.
    Cady, K.C., Bondy-Denomy, J., Heussler, G.E., Davidson, A.R.,O'Toole, G.A., 2012. The crispr/cas adaptive immune system of pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J. Bacteriol. 194, 5728-5738.
    Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K.,Madden, T.L., 2009. Blast+: architecture and applications. BMC Bioinf. 10, 421.
    Chatterjee, P., Jakimo, N.,Jacobson, J.M., 2018. Minimal pam specificity of a highly similar spcas9 ortholog. Sci. Adv. 4, eaau0766.
    Chyou, T.-Y.,Brown, C.M., 2019. Prediction and diversity of tracrrnas from type ii crispr-cas systems. RNA Biol. 16, 423-434.
    Common, J., Morley, D., Westra, E.R.,van Houte, S., 2019. Crispr-cas immunity leads to a coevolutionary arms race between streptococcus thermophilus and lytic phage. Philos. Trans. R. Soc. Lond. B Biol. Sci. 374, 20180098.
    Crooks, G.E., Hon, G., Chandonia, J.-M.,Brenner, S.E., 2004. Weblogo: a sequence logo generator. Genome Res. 14, 1188-1190.
    Deltcheva, E., Chylinski, K., Sharma, C.M., Gonzales, K., Chao, Y., Pirzada, Z.A., Eckert, M.R., Vogel, J.,Charpentier, E., 2011. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602-607.
    Demirci, S., Leonard, A., Essawi, K.,Tisdale, J.F., 2021. Crispr-cas9 to induce fetal hemoglobin for the treatment of sickle cell disease. Mol. Ther. Methods Clin. Dev. 23, 276-285.
    Doench, J.G., 2018. Am i ready for crispr? A user's guide to genetic screens. Nat. Rev. Genet. 19, 67-80.
    Esvelt, K.M., Mali, P., Braff, J.L., Moosburner, M., Yaung, S.J.,Church, G.M., 2013. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116-1121.
    Frangoul, H., Altshuler, D., Cappellini, M.D., Chen, Y.-S., Domm, J., Eustace, B.K., Foell, J., de la Fuente, J., Grupp, S., Handgretinger, R., et al., 2021. Crispr-cas9 gene editing for sickle cell disease and β-thalassemia. N. Engl. J. Med. 384, 252-260.
    Garneau, J.E., Dupuis, M.-v., Villion, M., Romero, D.A., Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P., Magadn, A.H.,Moineau, S., 2010. The crispr/cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67-71.
    Gleditzsch, D., Pausch, P., Mller-Esparza, H., zcan, A., Guo, X., Bange, G.,Randau, L., 2019. Pam identification by crispr-cas effector complexes: diversified mechanisms and structures. RNA Biol. 16, 504-517.
    Harrington, L.B., Burstein, D., Chen, J.S., Paez-Espino, D., Ma, E., Witte, I.P., Cofsky, J.C., Kyrpides, N.C., Banfield, J.F.,Doudna, J.A., 2018. Programmed DNA destruction by miniature crispr-cas14 enzymes. Science 362, 839-842.
    Heler, R., Samai, P., Modell, J.W., Weiner, C., Goldberg, G.W., Bikard, D.,Marraffini, L.A., 2015. Cas9 specifies functional viral targets during crispr-cas adaptation. Nature 519, 199-202.
    Hidalgo-Cantabrana, C., Goh, Y.J., Pan, M., Sanozky-Dawes, R.,Barrangou, R., 2019. Genome editing using the endogenous type i crispr-cas system in lactobacillus crispatus. Proc. Natl. Acad. Sci. U. S. A. 116, 15774-15783.
    Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A.,Charpentier, E., 2012. A programmable dual-rna-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821.
    Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., dek, A., Potapenko, A., et al., 2021. Highly accurate protein structure prediction with alphafold. Nature 596, 583-589.
    Kang, Y., Chu, C., Wang, F.,Niu, Y., 2019. Crispr/cas9-mediated genome editing in nonhuman primates. Dis. Model. Mech. 12, dmm039982.
    Karah, N., Samuelsen, r., Zarrilli, R., Sahl, J.W., Wai, S.N.,Uhlin, B.E., 2015. Crispr-cas subtype i-fb in acinetobacter baumannii: evolution and utilization for strain subtyping. PLoS One 10, e0118205.
    Kim, E., Koo, T., Park, S.W., Kim, D., Kim, K., Cho, H.-Y., Song, D.W., Lee, K.J., Jung, M.H., Kim, S., et al., 2017. In vivo genome editing with a small cas9 orthologue derived from campylobacter jejuni. Nat. Commun. 8, 14500.
    Komor, A.C., Badran, A.H.,Liu, D.R., 2017. Crispr-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20-36.
    Leenay, R.T., Maksimchuk, K.R., Slotkowski, R.A., Agrawal, R.N., Gomaa, A.A., Briner, A.E., Barrangou, R.,Beisel, C.L., 2016. Identifying and visualizing functional pam diversity across crispr-cas systems. Mol. Cell. 62, 137-147.
    Li, Z., Wang, X., Xu, D., Zhang, D., Wang, D., Dai, X., Wang, Q., Li, Z., Gu, Y., Ouyang, W., et al., 2020. Dnb-based on-chip motif finding: a high-throughput method to profile different types of protein-DNA interactions. Sci. Adv. 6, eabb3350.
    Makarova, K.S., Wolf, Y.I., Iranzo, J., Shmakov, S.A., Alkhnbashi, O.S., Brouns, S.J.J., Charpentier, E., Cheng, D., Haft, D.H., Horvath, P., et al., 2020. Evolutionary classification of crispr-cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67-83.
    Mao, Y., Botella, J.R., Liu, Y.,Zhu, J.-K., 2019. Gene editing in plants: progress and challenges. Natl. Sci. Rev. 6, 421-437.
    Mendoza, B.J.,Trinh, C.T., 2018. In silico processing of the complete crispr-cas spacer space for identification of pam sequences. Biotechnol. J. 13, e1700595.
    Mitrofanov, A., Ziemann, M., Alkhnbashi, O.S., Hess, W.R.,Backofen, R., 2022. Crisprtracrrna: robust approach for crispr tracrrna detection. Bioinformatics 38, ii42-ii48.
    Mu, Y., Zhang, C., Li, T., Jin, F.-J., Sung, Y.-J., Oh, H.-M., Lee, H.-G.,Jin, L., 2022. Development and applications of crispr/cas9-based genome editing in lactobacillus. Int. J. Mol. Sci. 23, 12852.
    Musunuru, K., Chadwick, A.C., Mizoguchi, T., Garcia, S.P., DeNizio, J.E., Reiss, C.W., Wang, K., Iyer, S., Dutta, C., Clendaniel, V., et al., 2021. In vivo crispr base editing of pcsk9 durably lowers cholesterol in primates. Nature 593, 429-434.
    Pausch, P., Al-Shayeb, B., Bisom-Rapp, E., Tsuchida, C.A., Li, Z., Cress, B.F., Knott, G.J., Jacobsen, S.E., Banfield, J.F.,Doudna, J.A., 2020. Crispr-casΦ from huge phages is a hypercompact genome editor. Science 369, 333-337.
    Pickar-Oliver, A.,Gersbach, C.A., 2019. The next generation of crispr-cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490-507.
    Pyne, M.E., Bruder, M.R., Moo-Young, M., Chung, D.A.,Chou, C.P., 2016. Harnessing heterologous and endogenous crispr-cas machineries for efficient markerless genome editing in clostridium. Sci. Rep. 6, 25666.
    Qin, Z., Yang, Y., Yu, S., Liu, L., Chen, Y., Chen, J.,Zhou, J., 2021. Repurposing the endogenous type i-e crispr/cas system for gene repression in gluconobacter oxydans wsh-003. ACS Synth. Biol. 10, 84-93.
    Ran, F.A., Cong, L., Yan, W.X., Scott, D.A., Gootenberg, J.S., Kriz, A.J., Zetsche, B., Shalem, O., Wu, X., Makarova, K.S., et al., 2015. m genome editing using staphylococcus aureus cas9. Nature 520, 186-191.
    Rybnicky, G.A., Fackler, N.A., Karim, A.S., Kpke, M.,Jewett, M.C., 2022. Spacer2pam: a computational framework to guide experimental determination of functional crispr-cas system pam sequences. Nucleic Acids Res. 50, 3523-3534.
    Sun, A., Li, C.-P., Chen, Z., Zhang, S., Li, D.-Y., Yang, Y., Li, L.-Q., Zhao, Y., Wang, K., Li, Z., et al., 2023. The compact casπ (cas12l) 'bracelet' provides a unique structural platform for DNA manipulation. Cell Res. 33, 229-244.
    Tang, L.C.,Gu, F., 2020. Next-generation crispr-cas for genome editing: focusing on the cas protein and pam. Yi Chuan 42, 236-249.
    Vale, P.F.,Little, T.J., 2010. Crispr-mediated phage resistance and the ghost of coevolution past. Proc. Biol. Sci. 277, 2097-2103.
    Walker, J.E., Lanahan, A.A., Zheng, T., Toruno, C., Lynd, L.R., Cameron, J.C., Olson, D.G.,Eckert, C.A., 2020. Development of both type i-b and type ii crispr/cas genome editing systems in the cellulolytic bacterium clostridium thermocellum. Metab. Eng. Commun. 10, e00116.
    Wang, J., Li, J., Zhao, H., Sheng, G., Wang, M., Yin, M.,Wang, Y., 2015. Structural and mechanistic basis of pam-dependent spacer acquisition in crispr-cas systems. Cell 163, 840-853.
    Wang, J.Y.,Doudna, J.A., 2023. Crispr technology: a decade of genome editing is only the beginning. Science 379, eadd8643.
    Xiao, Y., Ng, S., Nam, K.H.,Ke, A., 2017. How type ii crispr-cas establish immunity through cas1-cas2-mediated spacer integration. Nature 550, 137-141.
    Xu, C., Zhou, Y., Xiao, Q., He, B., Geng, G., Wang, Z., Cao, B., Dong, X., Bai, W., Wang, Y., et al., 2021. Programmable rna editing with compact crispr-cas13 systems from uncultivated microbes. Nat. Methods 18, 499-506.
    Xue, C.,Greene, E.C., 2021. DNA repair pathway choices in crispr-cas9-mediated genome editing. Trends Genet. 37, 639-656.
    Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., Volz, S.E., Joung, J., van der Oost, J., Regev, A., et al., 2015. Cpf1 is a single rna-guided endonuclease of a class 2 crispr-cas system. Cell 163, 759-771.
    Zhang, J., Zong, W., Hong, W., Zhang, Z.-T.,Wang, Y., 2018. Exploiting endogenous crispr-cas system for multiplex genome editing in clostridium tyrobutyricum and engineer the strain for high-level butanol production. Metab. Eng. 47, 49-59.
    Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-3415.
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