• T Tahir Institute of Molecular Biology & Biotechnology, University of Lahore, Lahore, Pakistan
  • Q Ali Institute of Biotechnology and Molecular Biology, The University of Lahore, Lahore
  • MS Rashid Institute of Molecular Biology & Biotechnology, University of Lahore, Lahore, Pakistan
  • A Malik Institute of Molecular Biology & Biotechnology, University of Lahore, Lahore, Pakistan



CRISPR-Cas9, endonucleases, geen editing, immune system, zinc finger, TALENS


Today we can use multiple of endonucleases for genome editing which has become very important and used in number of applications. We use sequence specific molecular scissors out of which, most important are mega nucleases, zinc finger nucleases, TALENS (Transcription Activator Like-Effector Nucleases) and CRISPR-Cas9 which is currently the most famous due to a number of reasons, they are cheap, easy to build, very specific in nature and their success rate in plants and animals is also high. Who knew that one day these CRISPR discovered as a part of immune system of bacteria will be this much worthwhile in the field of genetic engineering? This review interprets the science behind their mechanism and how several advancements were made with the passage of time to make them more efficient for the assigned job.


Download data is not yet available.


Abudayyeh O.O., Gootenberg J.S., Essletzbichler P., Han S., Joung J., Belanto J.J., Verdine V., Cox D.B.T., Kellner M.J., Regev A., et al. (2017). RNA targeting with CRISPR-Cas13. Nature. 550:280–284.

Abudayyeh O.O., Gootenberg J.S., Konermann S., Joung J., Slaymaker I.M., Cox D.B.T., Shmakov S., Makarova K.S., Semenova E., Minakhin L., et al. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573.

Ahi YS, Bangari DS, Mittal SK. (2011). Adenoviral vector immunity: its implications and circumvention strategies. CGT 11:307–20.

Ali, Z., Abul Faraj A., Li L., Ghosh N., Piatek M., Mahjoub A., Aouida M., et al. (2015). Efficient virus mediated genome editing in plants using the CRISPR/Cas9 system. Molecular Plant 8: 1288–1291.

Altpeter, F. , Springer N. M., Bartley L. E., Blechl A. E., Brutnell T. P., Citovsky V., Conrad L. J., et al. (2016). Advancing crop transformation in the era of genome editing. The Plant Cell 28: 1510–1520.

Antonio Regalado, China's CRISPR twins might have had their brains inadvertently enhanced, mit tech. rev (Feb. 21, 2019), (accessed Mar. 12, 2019)

Arnould, Sylvain; Perez, Christophe; Cabaniols, Jean-Pierre; Smith, Julianne; Gouble, Agnès; Grizot, Sylvestre; Epinat, Jean-Charles; Duclert, Aymeric; Duchateau, Philippe (2007-08-03). "Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells". Journal of Molecular Biology. 371 (1): 49–65.

Badis, G., Berger, M.F., Philippakis, A.A., et al. (2009). Diversity and complexity in DNA recognition by transcription factors. Science. 324(5935):1720–1723.

Bao A., Burritt D. J., Chen H., Zhou X., Cao D., Tran L. P. (2019). The CRISPR/Cas9 system and its applications in crop genome editing. Crit. Rev. Biotechnol. 39, 1–16.

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.

Barrangou, R., Marraffini, L.A. (2014). CRISPR-cas systems: Prokaryotes upgrade to adaptive immunity. Mol. Cell. 54:234–244.

Bassuk, A.G., Zheng, A., Li, Y., Tsang, S.H. and Mahajan, V.B., (2016). Precision medicine: genetic repair of retinitis pigmentosa in patient-derived stem cells. Scientific reports, 6, p.19969.

Bayat, H., Modarressi, M.H., Rahimpour, A. (2018). The conspicuity of CRISPR-Cpf1 system as a significant breakthrough in genome editing. Curr Microbiol. 75(1):107–115.

Bestor TH. (2000). Gene silencing as a threat to the success of gene therapy. J Clin Invest 105:409–11.

Beumer, K.J., Trautman, J.K., Bozas, A., Liu, J.L., Rutter, J., et al. (2008) Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc Natl Acad Sci U S A 105: 19821–19826

Bhat J.A., Ali S., Salgotra R.K., Mir Z.A., Dutta S., Jadon V., Tyagi A., Mushtaq M., Jain N., Singh P. (2017). Genomic selection in the era of next generation sequencing for complex traits in plant breeding. Front. Genet. 7:221.

Bikard D., Jiang W., Samai P., Hochschild A., Zhang F., Marraffini L.A. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41:7429–7437.

Bo W., Zhaohui Z., Huanhuan Z., Xia W., Binglin L., Lijia Y., Xiangyan H., Deshui Y., Xuelian Z., Chunguo W. (2019). Targeted Mutagenesis of NAC Transcription Factor Gene, OsNAC041, Leading to Salt Sensitivity in Rice. Rice Sci. 26:98–108.

Boch J., Scholze H., Schornack S., Landgraf A., Hahn S., Kay S., Lahaye T., Nickstadt A., Bonas U. (2009). Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. Science. 326:1509–1512.

Bolukbasi, M.F., Gupta, A., Oikemus, S., et al. (2015). DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat Methods 12:1150–6.

Bonamassa B, Hai L, Liu D. (2011). Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res 28:694–701.

Brown, M.T., Cooperm J.A. (1996). Regulation, substrates and functions of src. Biochim Biophys Acta. 1287:121–149.

Bruder M. R., Pyne M. E., Moo-Young M., Chung D. A., Chou C. P. (2016). Extending CRISPR-Cas9 technology from genome editing to transcriptional engineering in Clostridium. Appl. Environ. Microbiol. 82, 6109–6119.

Campa, C.C., Weisbach N.R., Santinha A.J., Incarnato D., and Platt R.J. (2019). Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nature Methods 16: 887–893.

Cannon, P., June, C (2011) Chemokine receptor 5 knockout strategies. Current Opinion in HIV and AIDS 6: 74–79.

Chen, S., Sanjana N.E., Zheng K., Shalem O., Shi X., Scott D.A., Song J., Pan J.Q., Weissleder R., Zhang F., et al. (2015). Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell. 160:1246–1260.

Chiou SH, Winters IP, Wang J, et al. (2015). Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev. 29:1576–1585.

Cho, S.W., Kim, S., Kim, Y., et al. (2014). Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24:132–41.

Choi, P.S., Meyerson, M. (2014). Targeted genomic rearrangements using CRISPR/Cas technology. Nat Commun. 5:3728.

Chu VT, Weber T, Wefers B, et al. (2015). Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–8.

Cohen S.N., Chang A.C.Y., Boyer H.W., Helling R.B. (1973). Construction of Biologically Functional Bacterial Plasmids In Vitro. Proc. Natl. Acad. Sci. USA.;70:3240–3244.

Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823.

Cradick, T.J., Fine, E.J., Antico, C.J., Bao, G. (2013). CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 41:9584–92.

Daya S, Berns KI. (2008). Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 21:583–93.

de Miguel Beriain I, del Cano AMM. (2018). Chapter 12 Gene editing in human embryos. A comment on the ethical issues involved. In: Soniewicka M , editor. The Ethics of Reproductive Genetics: Between Utility, Principles, and Virtues. Springer, Cham; 2018. pp. 173–183.

Dellomonaco C., Fava F., Gonzalez R. (2010). The path to next generation biofuels: Successes and challenges in the era of synthetic biology. Microb. Cell Fact. 9:3

Deyle DR, Russell DW. (2009). Adeno-associated virus vector integration. Curr Opin Mol Ther 11:442–7.

Dufossé L., Fouillaud M., Caro Y., Mapari S. A., Sutthiwong N. (2014). Filamentous fungi are large-scale producers of pigments and colorants for the food industry. Curr. Opin. Biotech. 26, 56–61.

Elbashir S.M., Harborth J., Lendeckel W., Yalcin A., Weber K., Tuschl T. (2001). Duplexes of 21 ± nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494–498.

Elferjani R., Soolanayakanahally R. (2018). Canola responses to drought, heat, and combined stress: shared and specific effects on carbon assimilation, seed yield, and oil composition. Front. Plant Sci. 9, 1224.

Endo A., Masafumi M., Kaya H., Toki S. (2016). Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci. Rep. 6:38169.

Epinat, Jean-Charles; Arnould, Sylvain; Chames, Patrick; Rochaix, Pascal; Desfontaines, Dominique; Puzin, Clémence; Patin, Amélie; Zanghellini, Alexandre; Pâques, Frédéric (2003-06-01). "A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells". Nucleic Acids Research. 31 (11): 2952–2962.

Follenzi A, Santambrogio L, Annoni A. (2007). Immune responses to lentiviral vectors. Curr Gene Ther 7:306–15.

Fonfara I., Richter H., BratoviÄ M., Le Rhun A., Charpentier E. (2016). The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 532:517–521.

Fu, Y., Foden, J.A., Khayter, C., et al. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–6.

Gabriel, R., Lombardo, A., Arens, A., Miller, J.C., Genovese, P., Kaeppel, C., Nowrouzi, A., Bartholomae, C.C., Wang, J., Friedman, G., et al. (2011). An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 29: 816–823.

Gao L., Cox D.B.T., Yan W.X., Manteiga J.C., Schneider M.W., Yamano T., Nishimasu H., Nureki O., Crosetto N., Zhang F. (2016). Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35:789–792.

Gao, L., Cox D. B. T., Yan W. X., Manteiga J. C., Schneider M. W., Yamano T., et al. (2017). Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792.

Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109:E2579–E2586.

Geurts, A.M., Cost, G.J., Freyvert, Y., Zeitler, B., Miller, J.C., et al. (2009) Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases. Science 325: 433.

Glass, Z. , Lee M., Li Y., and Xu Q.. 2018. Engineering the delivery system for CRISPR‐based genome editing. Trends in Biotechnology 36: 173–185.

Grissa, I., Vergnaud, G., Pourcel, C. (2007). The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinform. 8:1–10.

Guan Y, Ma Y, Li Q, et al. (2016). CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med 8:477–88.

Guilinger, J.P., Pattanayak, V., Reyon, D., Tsai, S.Q., Sander, J.D., Joung, J.K., Liu, D.R. (2014a). Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods 11: 429–435.

Halterman D., Guenthner J., Collinge S., Butler N., Douches D. (2015). Biotech Potatoes in the 21st Century: 20 years since the first biotech potato. Am. J. Potato 93: 1–20.

Hao Z., Su X. (2019). Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex. BMC Biotechnol. 19:2.

Horii T, Arai Y, Yamazaki M, et al. (2014). Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep 4:4513.

Hunter M. C., Smith R. G., Schipanski M. E., Atwood L. W., Mortensen D. A. (2017). Agriculture in 2050: recalibrating targets for sustainable intensification. Bioscience 67, 385–390.

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–1278

Ishino, Y., Shinagawam H., Makinom K., Amemura, M., Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433.

Jacobs J. Z., Ciccaglione K. M., Tournier V., Zaratiegui M. (2014). Implementation of the CRISPR-Cas9 system in fission yeast. Nat. Commun. 5:5344. 10.1038/ncomms6344

Jansen, R., van Emben, J.D.A., Gaastra, W. and Schouls L.M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43 1565–

Ji X. J., Ren L. J., Nie Z. K., Huang H., Ouyang P. K. (2014). Fungal arachidonic acid-rich oil: research, development and industrialization. Crit. Rew. Biotechnol. 34, 197–214.

Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31:233–239.

Jiang, F., Doudna J. A. (2017). CRISPR-Cas9 structures and mechanisms. Annu. Rev. Biophys. 46, 505–529.

Jiang, F., Doudna J. A. (2017). CRISPR-Cas9 structures and mechanisms. Annu. Rev. Biophys. 46, 505–529.

Jiang, W., Zhou H., Bi H., Fromm M., Yang B., and Weeks D. P. (2013). Demonstration of CRISPR/Cas9/sgRNA mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research 41: e188.

Jiang, W., Bikard, D., Cox, D., et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–9.

Jinek M, Chylinski K, Fonfara I, et al. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–21.

Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. (2014). Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res 42:e147.

Kaminski R., Chen Y., Fischer T., Tedaldi E., Napoli A., Zhang Y., Karn J., Hu W., Khalili K. (2016). Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Sci. Rep. 6:1–14.

Kaneko T., Sakuma T., Yamamoto T., Mashimo T. (2014). Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci. Rep. 4:1–5.

Kaur N., Alok A., Shivani, Kaur N., Pandey P., Awasthi P., et al. . (2018). CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. rasthali genome. Funct. Integr. Genomics 18, 89–99.

Kawamura, N., Nimura K., Nagano H., Yamaguchi S., Nonomura N., Kaneda Y. (2015) CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells. Oncotarget. 6:22361–22374.

Kennedy, E.M., Bassit L.C., Mueller H., Kornepati A.V.R., Bogerd H.P., Nie T., Chatterjee P., Javanbakht H., Schinazi R.F., Cullen B.R. (2015). Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology. 476:196–205.

Kerem B., Rommens J.M., Buchanan J.A., Markiewicz D., Cox T.K., Chakravarti A., Buchwald M., Tsui L.C. (1989). Identification of the cystic fibrosis gene: Genetic analysis. Science. 245:1073–1080.

Khatodia, S., Bhatotia K., Passricha N., Khurana S. M., Tuteja N. (2016). The CRISPR/Cas genome-editing tool: application in improvement of crops. Front. Plant Sci. 7:506.

Kim D, et al. (2016). Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 34:863–868.

Kim D., Kim D., Alptekin B., Budak H. (2017). CRISPR/Cas9 genome editing in wheat. Funct. Integr. Genomics. 18:31–41.

Kim H., Kim J.S. (2014). A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15:321–334.

Kim H., Kim S.T., Ryu J., Kang B.C., Kim J.S., Kim S.G. (2017). CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat. Commun. 8:14406.

Kim S.K., Seong W., Han G.H., Lee D.H., Lee S.G. (2017). CRISPR interference-guided multiplex repression of endogenous competing pathway genes for redirecting metabolic flux in Escherichia coli. Microb. Cell Fact. 16:188.

Kim, D., Bae, S., Park, J., et al. (2015). Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 12(3):237–243.

Kim, S., Kim, D., Cho, S.W., et al. (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–9.

Kim, Y.G., Cha, J., Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93: 1156–1160.

Kleinstiver, B.P., Pattanayak, V., Prew, M.S., et al. (2016). High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–5.

Kleinstiver, B.P., Prew, M.S., Tsai, S.Q., et al. (2015). Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol 33:1293–8.

Klimek-Chodacka M., Oleszkiewicz T., Lowder L.G., Qi Y., Baranski R. (2018). Efficient CRISPR/Cas9-based genome editing in carrot cells. Plant Cell Rep. 37:575–586.

Langner T., Kamoun S., Belhaj K. (2018). CRISPR Crops: Plant Genome Editing Toward Disease Resistance. Annu. Rev. Phytopathol. 56:479–512.

Lanzkron S., Carroll C.P., Haywood C., Jr. (2013). Mortality rates and age at death from sickle cell disease: U.S., 1979–2005. Public Health Rep.128:110–116.

Lee K, Conboy M, Park HM, et al. (2017). Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng 1:889–901.

Lee, H.J., Kim, E., Kim, J.S. (2010). Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res 20: 81–89.

Li A., Jia S., Yobi A., Ge Z., Sato S. J., Zhang C., et al. (2018a). Editing of an Alpha-Kafirin gene family increases, digestibility and protein quality in Sorghum. Plant Physiol. 177, 1425–1438.

Li B., Niu Y., Ji W., Dong Y. Strategies for the CRISPR-Based Therapeutics. Trends Pharmacol. Sci. 2020;41:55–65.

Li C., Ding L., Sun C.W., Wu L.C., Zhou D., Pawlik K.M., Khodadadi-Jamayran A., Westin E., Goldman F.D., Townes T.M. (2016). Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction. Sci. Rep. 6:1–10

Li H., Shen C. R., Huang C. H., Sung L. Y., Wu M. Y., Hu Y. C. (2016). CRISPR-Cas9 for the genome engineering of Cyanobacteria and succinate production. Metab. Eng. 38, 293–302.

Liang, P.P., Xu, Y.W., Zhang, X.Y., et al. (2015). CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 6(5):363–372.

Lin, S., Staahl, B.T., Alla, R.K., Doudna, J.A. (2014). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3:e04766.

Liu, G. , Li J., and Godwin I. D.. 2019. Genome editing by CRISPR/Cas9 in sorghum through biolistic bombardment In Zhao Z. Y. and Dahlberg J. [eds.], Sorghum, 169–183. Humana Press, New York, New York, USA

Liu, T., Shen J.K., Li Z., Choy E., Hornicek F.J., Duan Z. (2016). Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma. Cancer Lett. 373:109–118.

Ma H, Dang Y, Wu Y, et al. (2015). A CRISPR-based screen identifies genes essential for West-Nile-Virus-induced cell death. Cell Rep 12:673–83.

Ma, Y., Shen, B., Zhang, X., et al. (2014). Heritable multiplex genetic engineering in rats using CRISPR/Cas9. PLoS One 9:e89413

Macovei A., Sevilla N. R., Cantos C., Jonson G. B., Slamet-Loedin I., Èermák T., et al. (2018). Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus. Plant Biotechnol. J. 16 1918–1927.

Mahas A., Neal Stewart C., Mahfouz M.M. (2018). Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation. Biotechnol. Adv. 36:295–310.

Makarova, K.S., Grishin, N.V., Shabalina, S.A., Wolf, Y.I., and Koonin, E.V. (2006). A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct. 17

Mali P, Aach J, Stranges PB, et al. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–8.

Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M. (2013). RNA-guided human genome engineering via Cas9. Science 339:823–826.

Malnoy M., Viola R., Jung M.-H., Koo O.-J., Kim S., Kim J.-S., Velasco R., Nagamangala Kanchiswamy C. (2016). DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front. Plant Sci.7:1904.

Maruyama, T., Dougan, S.K., Truttmann, M.C., et al. (2015). Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 33:538–42.

Miao J., Guo D., Zhang J., Huang Q., Qin G., Zhang X., et al. (2013). Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res. 23 1233–1236.

Modrzejewski, D., Hartung F., Sprink T., Krause D., Kohl C., and Wilhelm R. (2019). What is the available evidence for the range of applications of genome editing as a new tool for plant trait modification and the potential occurrence of associated off target effects: A systematic map. Environmental Evidence 8: 27.

Moehle, E.A., Rock, J.M., Lee, Y.L., Jouvenot, Y., DeKelver, R.C., Gregory, P.D., Urnov, F.D., Holmes, MC. (2007). Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci 104: 3055–3060.

Mojica MJ, Juez G, Rodríguez-Valera F. 1993. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Mol Microbiol 9:613–621.

Moore, J.K., Haber J.E. (1996). Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:2164–2173.

Moscou, M.J., Bogdanove, A.J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science 326: 1501.

Mout R, Ray M, Yesilbag Tonga G, et al. (2017). Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano 11:2452–8.

Mussolino, C., Alzubi, J., Fine, E.J., Morbitzer, R., Cradick, T.J., Lahaye, T., Bao, G., Cathomen, T. (2014). TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res 42: 6762–6773.

Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31: 691-693.

Nishitani C., Hirai N., Komori S., Wada M., Okada K., Osakabe K., et al. (2016). Efficient genome editing in apple using a CRISPR/Cas9 system. Sci. Rep. 6:31481.

Osakabe Y., Osakabe K. Progress in Molecular Biology and Translational Science. Volume 149. Elsevier; Amsterdam, The Netherlands: 2017. Genome Editing to Improve Abiotic Stress Responses in Plants; pp. 99–109.

Papapetrou EP, Schambach A. (2016). Gene insertion into genomic safe harbors for human gene therapy. Mol Ther 24:678–84.

Paques, F., Duchateau, P. (2007). Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr Gene Ther 7: 49-66.

Pardo B., Gómez-González B., Aguilera A. (2009). DNA double-strand break repair: How to fix a broken relationship. Cell. Mol. Life Sci. 66:1039–1056.

Pattanayak, V., Ramirez, C.L., Joung, J.K., Liu, D.R. (2011). Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 8: 765–770.

Pessina S., Lenzi L., Perazzolli M., Campa M., Dalla Costa L., Urso S., Valè G., Salamini F., Velasco R., Malnoy M. (2016). Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine. Hortic. Res. 3:16016.

Platt R.J., Chen S., Zhou Y., Yim M.J., Swiech L., Kempton H.R., Dahlman J.E., Parnas O., Eisenhaure T.M., Jovanovic M., et al. (2014).CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell. 159:440–455.

Porteus MH, Carroll D. (2005). Gene targeting using zinc finger nucleases. Nat Biotechnol 23: 967–973.

Pyne M. E., Bruder M., Moo-Young M., Chung D. A., Chou C. P. (2014). Technical guide for genetic advancement of underdeveloped and intractable Clostridium. Biotechnol. Adv. 32, 623–641.

Pyott D. E., Sheehan E., Molnar A. (2016). Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants. Mol. Plant Pathol. 17 1276–1288.

Qi W., Zhu T., Tian Z., Li C., Zhang W., Song R. (2016). High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BMC Biotechnol.16:58.

Ramakrishna, S., Kwaku, Dad, A.B., Beloor, J., et al. (2014). Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020–7.

Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D.A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154: 1380–1389.

Raschmanova H., Weninger A., Glieder A., Kovar K., Vogl T. (2018). Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: current state and future prospects. Biotechnol. Adv. 36, 641–665.

Ricroch A., Clairand P., Harwood W. (2017). Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture. Emerg. Top. Life Sci. 1: 169–182.

Rodríguez-Leal D., Lemmon Z. H., Man J., Bartlett M. E., Lippman Z. B. (2017). Engineering quantitative trait variation for crop improvement by genome editing. Cell 171, 470–480.e8.

Rosinski-Chupin, I., Sauvage E., Fouet A., Poyart C., Glaser P. (2019). Conserved and specific features of Streptococcus pyogenes and Streptococcus agalactiae transcriptional landscapes. BMC Genomics 20, 236.

Samulski RJ, Muzyczka N. (2014). AAV-mediated gene therapy for research and therapeutic purposes. Annu Rev Virol 1:427–51.

Sánchez-León S., Gil-Humanes J., Ozuna C.V., Giménez M.J., Sousa C., Voytas D.F., Barro F. (2018). Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9. Plant Biotechnol. J. 16:902–910.

Sander, J. D., and Joung J. K.. 2014. CRISPR‐Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32: 347–355.

Savary S., Ficke A., Aubertot J. N., Hollier C. (2012). Crop losses due to diseases and their implications for global food production losses and food security. Food Secur. 4 519–537.

Savary S., Willocquet L., Pethybridge S.J., Esker P., McRoberts N., Nelson A. (2019). The global burden of pathogens and pests on major food crops. Nat. Ecol. Evol. 3:430–439.

Schwank G., Koo B.K., Sasselli V., Dekkers J.F., Heo I., Demircan T., Sasaki N., Boymans S., Cuppen E., Van Der Ent C.K., et al. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 13:653–658.

Shah T., Andleeb T., Lateef S., Noor M. A. (2018). Genome editing in plants: advancing crop transformation and overview of tools. Plant Physiol. Biochem. 131, 12–21.

Shan, Q. , Wang Y., Li J., Zhang Y., Chen K., Liang Z., Zhang K., et al. 2013. Targeted genome modification of crop plants using a CRISPR-Cas9 system. Nature Biotechnology 31: 686–688.

Shan, S. , Mavrodiev E. V., Li R., Zhang Z., Hauser B. A., Soltis P. S., Soltis D. E., and Yang B.. (2018). Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy. Molecular Ecology Resources 18: 1427–1443.

Shen B., Zhang W., Zhang J., Zhou J., Wang J., Chen L., Wang L., Hodgkins A., Iyer V., Huang X., and Skarnes W. C. (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Meth. 11, 399–402

Shen, B., Zhang, J., Wu, H., et al. (2013). Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 23:720–3.

Shi J., Gao H., Wang H., Lafitte H. R., Archibald R. L., Yang M., et al. (2017). ARGOS 8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnol. J. 15, 207–216.

Shukla, V.K., Doyon, Y., Miller, J.C., DeKelver, R.C., Moehle, E.A., et al. (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459: 437–441.

Slaymaker, I.M., Gao, L., Zetsche, B., et al. (2016). Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–8.

Smith. J., Grizot, S., Arnould, S., Duclert, A., Epinat, J.C., Chames, P., Prieto, J., Redondo, P., Blanco, F.J., Bravo, J., Montoya, G., Pâques, F., Duchateau, P. (2006). A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res. 34(22):e149.

Sternberg S. H., Redding S., Jinek M., Greene E. C., Doudna J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507: 62–67. 10.1038/nature13011.

Stoddard, B.L. (2005). Homing endonuclease structure and function. Q Rev Biophys 38: 49-95.

Stoddard, Barry L. (2006). "Homing endonuclease structure and function". Quarterly Reviews of Biophysics. 38 (1): 49-95.

Suda T, Gao X, Stolz DB, Liu D. (2007). Structural impact of hydrodynamic injection on mouse liver. Gene Ther 14:129–37.

Sun W, Ji W, Hall JM, et al. (2015). Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew Chem Int Ed Engl 54:12029–33.

Sun W, Jiang T, Lu Y, et al. (2014). Cocoon-like self-degradable DNA nanoclew for anticancer drug delivery. J Am Chem Soc 136:14722–5.

Sun Y., Jiao G., Liu Z., Zhang X., Li J., Guo X., Du W., Du J., Francis F., Zhao Y., et al. (2018). Generation of High-Amylose Rice through CRISPR/Cas9-Mediated Targeted Mutagenesis of Starch Branching Enzymes. Front. Plant Sci. 8:298.

Swiech L., Heidenreich M., Banerjee A., Habib N., Li Y., Trombetta J., Sur M., Zhang F. (2015). In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33:102–106.

Szostak J.W., Orr-Weaver T.L., Rothstein R.J., Stahl F.W. (1983). The double-strand-break repair model for recombination. Cell, 33:25–35.

Takeuchi, R., Choi, M., Stoddard, B.L. (2014). Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization. Proc Natl Acad Sci U S A. 111(11):4061-6.

Thomas V., Hartner F. S., Anton G. (2013). New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris. Curr. Opin. Biotech. 24, 1094–1101. 10.1016/j.copbio.2013.02.024

Thomazella D.P.D.T., Brail Q., Dahlbeck D., Staskawicz B.J. (2016). CRISPR-Cas9 mediated mutagenesis of a DMR6 ortholog in tomato confers broad-spectrum disease resistance. bioRxiv. 2016.

Thyme, S.B., Boissel, S.J., Arshiya, Quadri, S., Nolan, T., Baker, D.A., Park, R.U., Kusak, L., Ashworth, J., Baker, D. (2014). Reprogramming homing endonuclease specificity through computational design and directed evolution. Nucleic Acids Res. 42(4):2564-76

Torres, R., Martin, M.C., Garcia, A., Cigudosa, J.C., Ramirez, J.C., Rodriguez-Perales, S. (2014). Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system. Nat Commun 5: 3964

Trenberth K. E., Dai A., Van Der Schrier G., Jones P. D., Barichivich J., Briffa K. R., et al. (2014). Global warming and changes in drought. Nat. Clim. Change 4, 17.

Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S., Gregory, P.D. (2010). Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11: 636–646.

Valsalakumari J., Baby J., Bijin E., Constantine I., Manjila S., Pramod K. (2013). Novel gene delivery systems. Int. J. Pharm. Investig. 3:1.

van Regenmortel M.H., Mahy B.W. Desk Encyclopedia of Plant and Fungal Virology. Elsevier; San Diego, CA, USA: 2009.

Vartak, S.V., Raghavan, S.C. (2015). Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing. FEBS J 282:4289–94.

Voets O, Tielen F, Elstak E, et al. (2017). Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells. PLoS One 12:e0182974.

Waltz E. (2016). Gene-edited CRISPR mushroom escapes US regulation. Nature 532: 293.

Wang D., Samsulrizal N.H., Yan C., Allcock N.S., Craigon J., Blanco-Ulate B., Ortega-Salazar I., Marcus S.E., Bagheri H.M., Fons L.P. (2019). Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato. Plant Physiol. 179:544–557.

Wang F., Wang C., Liu P., Lei C., Hao W., Gao Y., Liu Y.-G., Zhao K. (2016). Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS ONE. 11:e0154027.

Wang L., Chen L., Li R., Zhao R., Yang M., Sheng J., et al. (2017). Reduced drought tolerance by CRISPR/Cas9-mediated SlMAPK3 mutagenesis in tomato plants. J. Agric. Food Chem. 65, 8674–8682.

Wang M, Zuris JA, Meng F, et al. (2016). Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc Natl Acad Sci USA 113:2868–73.

Wang S., Dong S., Wang P., Tao Y., Wang Y. (2017). Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 system. Appl. Environ. Microbiol. 83:e00233-17.

Wang W., Pan Q., He F., Akhunova A., Chao S., Trick H., et al. (2018). Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. CRISPR J. 1, 65–74.

Wang X., Wang Y., Wu X., Wang J., Wang Y., Qiu Z., Chang T., Huang H., Lin R.J., Yee J.K. (2015). Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33:175–178.

Wang Y., Cheng X., Shan Q., Zhang Y., Liu J., Gao C., Qiu J. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32:947.

Waters, C.A., Strande N.T., Pryor J.M., Strom C.N., Mieczkowski P., Burkhalter M.D., Oh S., Qaqish B.F., Moore D.T., Hendrickson E.A., et al. (2014). The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining. Nat. Commun. 5:4286.

Weeks, D.P., Spalding M. H., Yang B. (2016). Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnol. J. 14: 483–495.

Weirauch MT, Hughes TR. (2010). Conserved expression without conserved regulatory sequence: the more things change, the more they stay the same. Trends in Genetics. 26(2):66–74.

Wenzhi, J., Huanbin, Z., Honghao, B., Michael, F. and Bing, Y., Weeks Donald P. (2013): Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research, 41, pp.e188-e188.

White MK, Khalili K. (2016). CRISPR/Cas9 and cancer targets: future possibilities and present challenges. Oncotarget. 7:12305–12317.

Wolt J. D., Wang K., Yang B. (2016). The regulatory status of genome-edited crops. Plant Biotechnol. J. 14 510–518.

Woo, J. W. , Kim J., Kwon S. I., Corvalán C., Cho S. W., Kim H., Kim S. G., et al. (2015). DNA‐free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nature Biotechnology 33: 1162–1164.

Wu J.J., Du G.C., Chen J., Zhou J.W. (2015). Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli. Sci. Rep. 5:13477.

Wu Z, Yang H, Colosi P. (2010). Effect of genome size on AAV vector packaging. Mol Ther 18:80–6

Xie, K. , and Yang Y.. 2013. RNAguided genome editing in plants using a CRISPR-Cas9 system. Molecular Plant 6: 1975

Xu R., Yang Y., Qin R., Li H., Qiu C., Li L., et al. . (2016). Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice. J. Genet. Genomics 43, 529–532.

Xu, T., Li, Y., Shi, Z., Hemme, C.L., Li, Y., Zhu, Y., Van, Nostrand, J.D., He, Z., Zhou, J. (2015). Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase. Appl Environ Microbiol 81:4423–4431.

Xue W., Chen S., Yin H., Tammela T., Papagiannakopoulos T., Joshi N.S., Cai W., Yang G., Bronson R., Crowley D.G., et al. (2014). CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 514:380–384.

Yin H, Song CQ, Dorkin JR, et al. (2016). Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 34:328–33.

Yin H., Xue W., Chen S., Bogorad R.L., Benedetti E., Grompe M., Koteliansky V., Sharp P.A., Jacks T., Anderson D.G. (2013). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32:551–553.

Yu, C., Liu, Y., Ma, T., et al. (2015). Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16:142–7.

Zeisel, M.B., Lucifora J., Mason W.S., Sureau C., Beck J., Levrero M., Kann M., Knolle P.A., Benkirane M., Durantel D., et al. (2015). Towards an HBV cure: State-of-the-art and unresolved questions-report of the ANRS workshop on HBV cure. Gut. 64:1314–1326.

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 A., Liu Y., Wang F., Li T., Chen Z., Kong D., Bi J., Zhang F., Luo X., Wang J. (2019). Enhanced rice salinity tolerance via CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene. Mol. Breed. 39:47.

Zhang C., Meng X., Wei X., Lu L. (2016). Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet. Biol. 86, 47–57.

Zhang F., Wen Y., Guo X. (2014). CRISPR for genome editing: progress, implications and challenges. Hum. Mol. Genet. 23 40–46.

Zhang J., Zhang H., Botella J.R., Zhu J. (2018). Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties. J. Integr. Plant Biol. 60:369–375.

Zhang X., Lu G., Long W., Zou X., Li F., Nishio T. (2014). Recent progress in drought and salt tolerance studies in Brassica crops. Breed Sci. 64, 60–73.

Zhang Y, Ma X, Xie X, Liu Y-G. (2017). CRISPR/Cas9-based genome editing in plants. Prog Mol Biol Transl Sci 149:133–50.

Zhang Y., Massel K., Godwin I. D., Gao C. (2018b). Applications and potential of genome editing in crop improvement. Genome Biol. 19, 210.

Zhang Y., Yu L.C. (2008). Single-cell microinjection technology in cell biology. BioEssays. 30:606–610.

Zhen S, Hua L, Liu YH, et al. (2015). Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus. Gene Ther 22:404–12.

Zhou Y, Zhu S, Cai C, et al. (2014). High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509:487–91.

Zhu M., Monroe J. G., Suhail Y., Villiers F., Mullen J., Pater D., et al. (2016). Molecular and systems approaches towards drought-tolerant canola crops. New Phytol. 210, 1169–1189.




How to Cite

Tahir, T., Ali, Q., Rashid, M., & Malik, A. (2020). THE JOURNEY OF CRISPR-CAS9 FROM BACTERIAL DEFENSE MECHANISM TO A GENE EDITING TOOL IN BOTH ANIMALS AND PLANTS. Biological and Clinical Sciences Research Journal, 2020(1).



Review Articles

Most read articles by the same author(s)

1 2 3 4 5 > >>