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Genome sequence of detection for fewer infertility and development in the genome variable methods has opened up the incredible effects of elevating characteristics of the organisms. Pathway to new genome mutations, for example zinc finger nucleases (ZFNAS), transcription activator-like effectors nucleases (TALENS) records have made it easier molecular experts to focus more on any quality of interest. However, these processes are expensive and tedious as they add difficult steps that require protein synthesis. Not at all like the instruments that change things, modifications of CRISPR/ cas9 type includes basic planning techniques and integration strategies, with the same Cas9 has been found clearly assessable for use by various targeted RNAs at various locations in the genome. After confirmation of the concept show in production plants including the critical CRISPR/Cas9 module several Cas9 proteins types have been modified which are being used in production plants to improve quality and yield (e.g Nmcas9, SaCas9 Snd stCas9). The study summarizes the sum of the most accessible methods investing biotechnologists to achieve plant development using CRISPR/Cas9 based genome modification devices and further introduces the experiments in which CRISPR/Cas9 was used in improving the tolerance for biotic and abiotic stress. The use of these methods leads to in the development of non transgenic or non-modified plants with appropriate potential that can add to the potential expansion under biotic and abiotic stress conditions.


CRISPR, TALEN, quantitative traits loci, biotic stress, abiotic stress.

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Adli M. The CRISPR tool kit for genome editing and beyond. Nature Communications. 2018;9(1):1-13.

Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances. 2015; 33(1):41-52.

Cho SW, Kim S, Kim JM, Kim J-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature Biotechnology. 2013; 31(3):230-232.

Javied MA, Ashfaq N, Haider MA, Fatima F, Ali Q, et al. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) using dmo gene for enhanced tolerance against dicamba pesticide. Biological and Clinical Sciences Research Journal. 2021;2021(1):e009.

Akram A, Arshad K, Hafeez MN. Cloning and expression of universal stress protein 2 (USP2) gene in Escherichia coli. Biological and Clinical Sciences Research Journal. 2021;2021(1):e002.

Ejaz RM, Ahmad S, M Ali, H Choudhry, S Anti-biofilm potential of menthol purified from Mentha piperita L. (Mint). Biol Clin Sci Res J. 2020;202(e037).

Chandrasegaran S, Carroll D. Origins of programmable nucleases for genome engineering. Journal of Molecular Biology. 2016;428(5):963-989.

Jiang W, Marraffini LA. CRISPR-Cas: new tools for genetic manipulations from bacterial immunity systems. Annual Review of Microbiology. 2015; 69209-228.

Horvath P, Romero DA, Coûté-Monvoisin A-C, Richards M, Deveau H, et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. Journal of Bacteriology. 2008;190(4):1401-1412.

Balqees N, Ali Q, Malik A. Genetic evaluation for seedling traits of maize and wheat under biogas wastewater, sewage water and drought stress conditions. Biol Clin Sci Res J. 2020;2020:(e038).

Danish P, Ali QH, MM, Malik A. Antifungal and antibacterial activity of aloe vera plant extract. Biol Clin Sci Res J. 2020;2020e003.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.

Komor AC, Badran AH, Liu DR. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017;168(1-2):20-36.

Liang P, Xu Y, Zhang X, Ding C, Huang R, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & cell. 2015;6(5):363-372.

Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-Cas systems. Current Opinion in Microbiology. 2017;3767-78.

Ghafoor M, Ali Q, Malik A. Effects of salicylic acid priming for salt stress tolerance in wheat. Biol Clin Sci Res J. 2020;2020:(e024).

Luo ML, Leenay RT, Beisel CL. Current and future prospects for CRISPR‐ based tools in bacteria. Biotechnology and Bioengineering. 2016;113(5):930-943.

Maeder ML, Gersbach CA. Genome-editing technologies for gene and cell therapy. Molecular Therapy. 2016;24(3): 430-446.

Naseem S, Ali Q, Malik A. Evaluation of maize seedling traits under salt stress. Biological and Clinical Sciences Research Journal. 2020;2020(e025).

Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature Biotechnology. 2014;32(7):677-683.

Plessis A, Perrin A, Haber J, Dujon B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics. 1992;130(3):451-460.

Asif S, Ali Q, Malik A. Evaluation of salt and heavy metal stress for seedling traits in wheat. Biol Clin Sci Res J. 2020;2020: e005.

Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152(5):1173-1183.

Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E. Chopchop: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Research. 2014;42(W1):W401-W407.

Bashir A, Ali Q, Rashid M, Malik A. CRISPR/CAS9 in genome editing: A nature gifted molecualr tool. Biol Clin Sci Res J. 2020;2020(e018).

Silva G, Poirot L, Galetto R, Smith J, Montoya G, et al. Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Current Gene Therapy. 2011; 11(1):11-27.

Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology. 2014;32(4):347-355.

Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nature Biotechnology. 2016;34(9): 933-941.

Bondy-Denomy J, Davidson AR. To acquire or resist: the complex biological effects of CRISPR–Cas systems. Trends in Microbiology. 2014;22(4):218-225.

Chang HH, Pannunzio NR, Adachi N, Lieber MR. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nature Reviews Molecular Cell Biology. 2017;18(8):495.

Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols. 2013;8(11):2180-2196.

Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. 2009;155(3):733-740.

Nelles DA, Fang MY, O’Connell MR, Xu JL, Markmiller SJ, et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell. 2016;165(2):488-496.

Pederson T. Repeated TALEs: visualizing DNA sequence localization and chromosome dynamics in live cells. Nucleus. 2014;5(1):28-31.

Selle K, Klaenhammer TR, Barrangou R. CRISPR-based screening of genomic island excision events in bacteria. Proceedings of the National Academy of Sciences. 2015;112(26):8076-8081.

Yaqoob SFN, Khan S, Ali Q, Hafeez MM, Malik A. Begomoviruses and betasatellites associated with CLCuD. Biol Clin Sci Res J. 2020;2020:e002.

Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 2015;348(6230): 62-68.

Ali M, Rafique F, Ali Q, Malik A. Genetic modification for salt and drought tolerance in plants through SODERF3. Biological and Clinical Sciences Research Journal. 2020;2020:(e022).

Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012; 482(7385):331-338.

Thanisch K, Schneider K, Morbitzer R, Solovei I, Lahaye T, et al. Targeting and tracing of specific DNA sequences with dTALEs in living cells. Nucleic Acids Research. 2014;42(6):e38-e38.

Wang H, La Russa M, Qi LS. CRISPR/Cas9 in genome editing and beyond. Annual Review of Biochemistry. 2016;85227-264.

Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Molecular Cell. 2015;60(3):385-397.

Choulika A, Perrin A, Dujon B, Nicolas J-F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Molecular and Cellular Biology. 1995; 15(4):1968-1973.

Khalil R, Ali QHM, Malik A. Phenolic acid profiling by RP-HPLC: evaluation of antibacterial and anticancer activities of Conocarpus erectus plant extracts. Biol Clin Sci Res J. 2020;2020e010.

Khalil R, Ali QHM, Malik A. Phytochemical activities of Conocarpus erectus: An overview. Biol Clin Sci Res J. 2020;2020e008.

Nazir MI II, Danish P, Ahmad S, Ali Q, Malik A. Potential of water hyacinth (Eichhornia crassipes L.) for phytoremediation of heavy metals from waste water Biol Clin Sci Res J. 2020;2020e006.

Yousef F, Shafique F, Ali Q, Malik A. Effects of salt stress on the growth traits of chickpea (Cicer arietinum L.) and pea (Pisum sativum L.) seedlings. Biol Clin Sci Res J. 2020;2020:(e029).

Rudin N, Sugarman E, Haber JE. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics. 1989; 122(3):519-534.

Staals RH, Zhu Y, Taylor DW, Kornfeld JE, Sharma K, et al. RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Molecular Cell. 2014;56(4):518-530.

Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nature Reviews Genetics. 2016;17(5):300-312.

Wei W, Ba Z, Gao M, Wu Y, Ma Y, et al. A role for small RNAs in DNA double-strand break repair. Cell. 2012;149(1):101-112.

Nawaz A, Haseeb A, Malik H, Ali Q, Malik A. Genetic association among morphological traits of Zea mays seedlings under salt stress. Biol Clin Sci Res J. 2020;2020:(e021).

Yin H, Song C-Q, Dorkin JR, Zhu LJ, Li Y, et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nature Biotechnology. 2016;34(3):328- 333.

Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-771.

Tahir T, Ali Q, Rashid M, Malik A. The journey of CRISPR-Cas9 from bacterial defense mechanism to a gene editing tool in both animals and plants. Biol Clin Sci Res J. 2020;e017.

Muqadas S, Ali Q, Malik A. Genetic association among seedling traits of Zea mays under multiple stresses of salts, heavy metals and drought. Biological and Clinical Sciences Research Journal. 2020;2020: (e026).

Cong L, Ran FA, Cox D, Lin S, Barretto R, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-823.

Hameed B, Ali Q, Hafeez M, A M. Antibacterial and antifungal activity of fruit, seed and root extracts of Citrullus colocynthis plant. Biol Clin Sci Res J. 2020;2020:(e033).

Shafique F, Ali Q, Malik A. Effects of heavy metal toxicity on maize seedlings growth traits. Biol Clin Sci Res J. 2020;2020:(e027).

Shafique F, Ali Q, Malik A. Effects of water deficit on maize seedlings growth traits. Biol Clin Sci Res J, (2020); 2020(e028).

Künne T, Kieper SN, Bannenberg JW, Vogel AI, Miellet WR, et al. Cas3-derived target DNA degradation fragments fuel primed CRISPR adaptation. Molecular Cell. 2016;63(5):852-864.

Miller J, McLachlan A, Klug A. Repetitive zinc‐binding domains in the protein transcription factor IIIA from Xenopus oocytes. The EMBO Journal. 1985;4(6): 1609-1614.

Ashfaq F, Ali Q, Haider MA, Hafeez MM, Malik A. Therapeutic activities of garlic constituent phytochemicals. Biological and Clinical Sciences Research Journal. 2021;2021(1):e007.

Rees HA, Komor AC, Yeh W-H, Caetano-Lopes J, Warman M, et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nature Communications. 2017;8(1):1-10.

Samai P, Pyenson N, Jiang W, Goldberg GW, Hatoum-Aslan A, et al. Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity. Cell. 2015;161(5):1164-1174.

Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nature Protocols. 2014;9(10):2395-2410.

Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4): 910-918.

Gil‐Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, et al. High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. The Plant Journal. 2017;89(6):1251-1262.

Mali P, Yang L, Esvelt KM, Aach J, Guell M, et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-826.

Iqra L, Rashid M, Ali Q, Latif I, Malik A. Genetic variability for salt tolerance in wheat. Biol Clin Sci Res J. 2020; 2020:(e016).

Haseeb A, Nawaz A, Rao M, Ali Q, Malik A. Genetic variability and association among seedling traits of Zea mays under drought stress conditions. Biological and Clinical Sciences Research Journal. 2020; 2020:(e020).

Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014;513(7519):569-573.

Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151(8):2551-2561.

Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008;321(5891):960-964.

Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology. 2008;190(4):1390-1400.

Masood M, Ahsan M, Sadaqat HA, F. Screening of maize (Zea mays L.) inbred lines under water deficit conditions. Biol Clin Sci Res J. 2020;2020:e007.

Gao W, Long L, Tian X, Xu F, Liu J, et al. Genome editing in cotton with the CRISPR/Cas9 system. Frontiers in Plant Science. 2017;81364.

Chen X, Lu X, Shu N, Wang S, Wang J, et al. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Scientific Reports. 2017;7(1):1-7.

Long L, Guo D-D, Gao W, Yang W-W, Hou L-P, et al. Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression. Plant Methods. 2018;14(1):1-9.

Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA. Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology. 2015;15(1):1-10.

Chilcoat D, Liu Z-B, Sander J. Use of CRISPR/Cas9 for crop improvement in maize and soybean. Progress in Molecular Biology and Translational Science. 2017;14927-46.

Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nature Biotechnology. 2017;35(5):441-443.

Pan C, Ye L, Qin L, Liu X, He Y, et al. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific Reports. 2016;6(1):1-9.

Dahan‐Meir T, Filler‐Hayut S, Melamed‐Bessudo C, Bocobza S, Czosnek H, et al. Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system. The Plant Journal. 2018;95(1):5-16.

Li X, Wang Y, Chen S, Tian H, Fu D, et al. Lycopene is enriched in tomato fruit by CRISPR/Cas9-mediated multiplex genome editing. Frontiers in Plant Science. 2018;9559.

Wang R, da Rocha Tavano EC, Lammers M, Martinelli AP, Angenent GC, et al. Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis. Scientific Reports. 2019;9(1):1-10.

Wang S, Zhang S, Wang W, Xiong X, Meng F, et al. Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Reports. 2015;34(9): 1473-1476.

Nakayasu M, Akiyama R, Lee HJ, Osakabe K, Osakabe Y, et al. Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene. Plant Physiology and Biochemistry. 2018;13170-77.

Makhotenko A, Khromov A, Snigir E, Makarova S, Makarov V, et al. Functional analysis of coilin in virus resistance and stress tolerance of potato Solanum tuberosum using CRISPR-Cas9 editing; Springer. 2019;88-91.

Zhang F, LeBlanc C, Irish VF, Jacob Y. Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter. Plant Cell Reports. 2017;36(12):1883-1887.

Jia H, Orbović V, Wang N. CRISPR‐LbCas12a‐mediated modification of citrus. Plant Biotechnology Journal. 2019;17(10):1928-1937.

Goulin EH, Galdeano DM, Granato LM, Matsumura EE, Dalio RJD, et al. RNA interference and CRISPR: Promising approaches to better understand and control citrus pathogens. Microbiological Research. 2019;2261-9.

Sun L, Ke F, Nie Z, Wang P, Xu J. Citrus genetic engineering for disease resistance: past, present and future. International Journal of Molecular Sciences. 2019; 20(21):5256.

Nakajima I, Ban Y, Azuma A, Onoue N, Moriguchi T, et al. CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS One. 2017;12(5):e0177966.

Ren C, Liu X, Zhang Z, Wang Y, Duan W, et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Scientific Reports. 2016; 6(1):1-9.

Jaganathan D, Ramasamy K, Sellamuthu G, Jayabalan S, Venkataraman G. CRISPR for crop improvement: an update review. Frontiers in Plant Science. 2018;9985.

Ahmad S, Wei X, Sheng Z, Hu P, Tang S. CRISPR/Cas9 for development of disease resistance in plants: recent progress, limitations and future prospects. Briefings in Functional Genomics. 2020;19(1):26-39.

Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant. 2015;8(8):1274-1284.

Jiang W, Zhou H, Bi H, Fromm M, Yang B, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research. 2013;41(20):e188-e188.

Tang T, Yu X, Yang H, Gao Q, Ji H, et al. Development and validation of an effective CRISPR/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species. Frontiers in Plant Science. 2018;91533.

Waltz E. With a free pass, CRISPR-edited plants reach market in record time. Nature Publishing Group; 2018.