Abstract
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized transgene-free and multiplex genome editing. In this review, we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.
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Ali Z, Abul-faraj A, Li L, Ghosh N, Piatek M, Mahjoub A, Aouida M, Piatek A, Baltes NJ, Voytas DF, Dinesh-Kumar S, Mahfouz MM (2015) Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant 8:1288–1291. https://doi.org/10.1016/j.molp.2015.02.011
Baek K, Kim DH, Jeong J, Sim SJ, Melis A, Kim J-S, Jin E, Bae S (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep 6:30620. https://doi.org/10.1038/srep30620
Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF (2014) DNA replicons for plant genome engineering. Plant Cell 26:151–163. https://doi.org/10.1105/tpc.113.119792
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712. https://doi.org/10.1126/science.1138140
Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52. https://doi.org/10.1016/j.biotechadv.2014.12.006
Campa CC, Weisbach NR, Santinha AJ, Incarnato D, Platt RJ (2019) Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat Methods 16:887–893. https://doi.org/10.1038/s41592-019-0508-6
Čermák T, Curtin SJ, Gil-Humanes J, Čegan R, Kono TJY, Konečná E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217. https://doi.org/10.1105/tpc.16.00922
Chen G, Abdeen AA, Wang Y, Shahi PK, Robertson S, Xie R, Suzuki M, Pattnaik BR, Saha K, Gong S (2019) A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing. Nat Nanotechnol 14:974–980. https://doi.org/10.1038/s41565-019-0539-2
Cho SW, Lee J, Carroll D, Kim J-S, Lee J (2013) Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195:1177–1180. https://doi.org/10.1534/genetics.113.155853
Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim J-S (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24:132–141. https://doi.org/10.1101/gr.162339.113
Cody WB, Scholthof HB, Mirkov TE (2017) Multiplexed gene editing and protein overexpression using a tobacco mosaic virus viral vector. Plant Physiol 175:23–35. https://doi.org/10.1104/pp.17.00411
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143
Cunningham FJ, Goh NS, Demirer GS, Matos JL, Landry MP (2018) Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol 36:882–897. https://doi.org/10.1016/j.tibtech.2018.03.009
Dahlman JE, Abudayyeh OO, Joung J, Gootenberg JS, Zhang F, Konermann S (2015) Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat Biotechnol 33:1159–1161. https://doi.org/10.1038/nbt.3390
Decaestecker W, Andrade Buono R, Pfeiffer M, Vangheluwe N, Jourquin J, Karimi M, van Isterdael G, Beeckman T, Nowack MK, Jacobs TB (2019) CRISPR-TSKO: a technique for efficient mutagenesis in specific cell types, tissues, or organs in Arabidopsis. Plant Cell. https://doi.org/10.1105/tpc.19.00454
Demirer GS, Zhang H, Matos JL, Goh NS, Cunningham FJ, Sung Y, Chang R, Aditham AJ, Chio L, Cho M-J, Staskawicz B, Landry MP (2019) High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat Nanotechnol 14:456–464. https://doi.org/10.1038/s41565-019-0382-5
Ding D, Chen K, Chen Y, Li H, Xie K (2018) Engineering introns to express RNA guides for Cas9- and Cpf1-mediated multiplex genome editing. Mol Plant 11:542–552. https://doi.org/10.1016/j.molp.2018.02.005
Dong F, Xie K, Chen Y, Yang Y, Mao Y (2017) Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells. Biochem Biophys Res Commun 482:889–895. https://doi.org/10.1016/j.bbrc.2016.11.129
Doyle C, Higginbottom K, Swift TA, Winfield M, Bellas C, Benito-Alifonso D, Fletcher T, Galan MC, Edwards K, Whitney HM (2019) A simple method for spray-on gene editing in planta. bioRxiv. https://doi.org/10.1101/805036
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. https://doi.org/10.1038/srep38169
Ferreira R, Skrekas C, Nielsen J, David F (2018) Multiplexed CRISPR/Cas9 genome editing and gene regulation using Csy4 in Saccharomyces cerevisiae. ACS Synth Biol 7:10–15
Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405. https://doi.org/10.1016/j.tibtech.2013.04.004
Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56:343–349. https://doi.org/10.1111/jipb.12152
Gao Y, Zhang Y, Zhang D, Dai X, Estelle M, Zhao Y (2015) Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development. Proc Natl Acad Sci USA 112:2275–2280. https://doi.org/10.1073/pnas.1500365112
Gao Z, Herrera-Carrillo E, Berkhout B (2019) A single H1 promoter can drive both guide RNA and endonuclease expression in the CRISPR-Cas9 system. Mol Ther Nucleic Acids 14:32–40. https://doi.org/10.1016/j.omtn.2018.10.016
Hampf M, Gossen M (2007) Promoter crosstalk effects on gene expression. J Mol Biol 365:911–920. https://doi.org/10.1016/j.jmb.2006.10.009
Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358. https://doi.org/10.1126/science.1192272
He Y, Zhang T, Yang N, Xu M, Yan L, Wang L, Wang R, Zhao Y (2017) Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing. J Genet Genomics 44:469–472. https://doi.org/10.1016/j.jgg.2017.08.003
Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH (2015) Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33:985–989. https://doi.org/10.1038/nbt.3290
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827
Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:e188–e188. https://doi.org/10.1093/nar/gkt780
Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP (2014) Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryot Cell 13:1465–1469. https://doi.org/10.1128/EC.00213-14
Jing X, Xie B, Chen L, Zhang N, Jiang Y, Qin H, Wang H, Hao P, Yang S, Li X (2018) Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing. Nucleic Acids Res 46:e90–e90. https://doi.org/10.1093/nar/gky433
Katayama T, Tanaka Y, Okabe T, Nakamura H, Fujii W, Kitamoto K, Maruyama J (2016) Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol Lett 38:637–642. https://doi.org/10.1007/s10529-015-2015-x
Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJK, Ebrahimkhani MR, Collins JJ, Weiss R, Church G (2015) Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods 12:1051–1054. https://doi.org/10.1038/nmeth.3580
Kim S, Kim D, Cho SW, Kim J, Kim J-S (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019. https://doi.org/10.1101/gr.171322.113
Knapp DJHF, Michaels YS, Jamilly M, Ferry QRV, Barbosa H, Milne TA, Fulga TA (2019) Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression. Nat Commun 10:1490. https://doi.org/10.1038/s41467-019-09148-3
Kurata M, Wolf NK, Lahr WS, Weg MT, Kluesner MG, Lee S, Hui K, Shiraiwa M, Webber BR, Moriarity BS (2018) Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays. PLoS ONE 13:e0198714–e0198714. https://doi.org/10.1371/journal.pone.0198714
Lee RTH, Ng ASM, Ingham PW (2016) Ribozyme mediated gRNA generation for in vitro and in vivo CRISPR/Cas9 mutagenesis. PLoS ONE 11:e0166020–e0166020. https://doi.org/10.1371/journal.pone.0166020
Lee K, Mackley VA, Rao A, Chong AT, Dewitt MA, Corn JE, Murthy N (2017) Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife 6:e25312. https://doi.org/10.7554/eLife.25312
Li J, Aach J, Norville JE, Mccormack M, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated plant genome editing via guide RNA/Cas9. Nat Biotechnol 31:688–691. https://doi.org/10.1038/nbt.2654.Multiplex
Li Y, Bolinger J, Yu Y, Glass Z, Shi N, Yang L, Wang M, Xu Q (2019) Intracellular delivery and biodistribution study of CRISPR/Cas9 ribonucleoprotein loaded bioreducible lipidoid nanoparticles. Biomater Sci 7:596–606. https://doi.org/10.1039/C8BM00637G
Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261. https://doi.org/10.1038/ncomms14261
Liu W, Stewart CN (2016) Plant synthetic promoters and transcription factors. Curr Opin Biotechnol 37:36–44. https://doi.org/10.1016/j.copbio.2015.10.001
Liu W, Mazarei M, Rudis MR, Fethe MH, Peng Y, Millwood RJ, Schoene G, Burris JN, Stewart CN Jr (2013) Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants. Plant Biotechnol J 11:43–52. https://doi.org/10.1111/pbi.12005
Liu Q, Shi X, Song L, Liu H, Zhou X, Wang Q, Zhang Y, Cai M (2019) CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris. Microb Cell Fact 18:144. https://doi.org/10.1186/s12934-019-1194-x
Lowder L, Zhang D, Baltes NJ, Paul JW, Tang X, Zheng X, Voytas DF, Hsieh T-F, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169:971–985. https://doi.org/10.1104/pp.15.00636
Lowder L, Malzahn A, Qi Y (2016) Rapid evolution of manifold CRISPR systems for plant genome editing. Front Plant Sci 7:1683. https://doi.org/10.3389/fpls.2016.01683
Lowder LG, Zhou J, Zhang Y, Malzahn A, Zhong Z, Hsieh T, Voytas DF, Zhang Y, Qi Y (2018) Robust transcriptional activation in plants using systems. Mol Plant 11:245–256. https://doi.org/10.1016/j.molp.2017.11.010
Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y-G (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284. https://doi.org/10.1016/j.molp.2015.04.007
Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NGA, van den Broek M, Daran-Lapujade P, Pronk JT, van Maris AJA, Daran J-MG (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res. https://doi.org/10.1093/femsyr/fov004
Mikami M, Toki S, Endo M (2017) In planta processing of the SpCas9-gRNA complex. Plant Cell Physiol 58:1857–1867. https://doi.org/10.1093/pcp/pcx154
Minkenberg B, Wheatley M, Yang Y (2017) CRISPR/Cas9-enabled multiplex genome editing and its application. In: Weeks DP, Yang B (eds) Gene editing in plants, vol 149, PMBTS. Academic Press, UK, pp 111–132
Moon SB, Kim DY, Ko J-H, Kim J-S, Kim Y-S (2019) Improving CRISPR genome editing by engineering guide RNAs. Trends Biotechnol 37:870–881. https://doi.org/10.1016/j.tibtech.2019.01.009
Mu W, Zhang Y, Xue X, Liu L, Wei X, Wang H (2019) 5′ capped and 3′ polyA-tailed sgRNAs enhance the efficiency of CRISPR-Cas9 system. Protein Cell 10:223–228. https://doi.org/10.1007/s13238-018-0552-5
Nekrasov V, Staskawicz B, Weigel D, Jones JDG, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691
Nie L, Das TM, Wang Y, Su Q, Zhao Y, Feng Y (2010) Regulation of U6 promoter activity by transcriptional interference in viral vector-based RNAi. Genomics Proteomics Bioinformatics 8:170–179. https://doi.org/10.1016/S1672-0229(10)60019-8
Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK (2014) Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell 54:698–710. https://doi.org/10.1016/j.molcel.2014.04.022
Nødvig CS, Hoof JB, Kogle ME, Jarczynska ZD, Lehmbeck J, Klitgaard DK, Mortensen UH (2018) Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genet Biol 115:78–89. https://doi.org/10.1016/j.fgb.2018.01.004
Peng R, Lin G, Li J (2016) Potential pitfalls of CRISPR/Cas9-mediated genome editing. FEBS J 283:1218–1231. https://doi.org/10.1111/febs.13586
Peterson BA, Haak DC, Nishimura MT, Teixeira PJPL, James SR, Dangl JL, Nimchuk ZL (2016) Genome-wide assessment of efficiency and specificity in CRISPR/Cas9 mediated multiple site targeting in arabidopsis. PLoS ONE 11:e0162169–e0162169. https://doi.org/10.1371/journal.pone.0162169
Poe AR, Wang B, Sapar ML, Ji H, Li K, Onabajo T, Fazliyeva R, Gibbs M, Qiu Y, Hu Y, Han C (2019) Robust CRISPR/Cas9-mediated tissue-specific mutagenesis reveals gene redundancy and perdurance in Drosophila. Genetics 211:459–472. https://doi.org/10.1534/genetics.118.301736
Port F, Bullock SL (2016) Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods 13:852–854. https://doi.org/10.1038/nmeth.3972
Port F, Chen H-M, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci USA 111:E2967–E2976. https://doi.org/10.1073/pnas.1405500111
Qin W, Liang F, Feng Y, Bai H, Yan R, Li S, Lin S (2015) Expansion of CRISPR/Cas9 genome targeting sites in zebrafish by Csy4-based RNA processing. Cell Res 25:1074–1077. https://doi.org/10.1038/cr.2015.95
Ren Q, Zhong Z, Wang Y, You Q, Li Q, Yuan M, He Y, Qi C, Tang X, Zheng X, Zhang T, Qi Y, Zhang Y (2019) Bidirectional promoter-based CRISPR-Cas9 systems for plant genome editing. Front Plant Sci 10:1173
Ryan OW, Skerker JM, Maurer MJ, Li X, Tsai JC, Poddar S, Lee ME, DeLoache W, Dueber JE, Arkin AP, Cate JHD (2014) Selection of chromosomal DNA libraries using a multiplex CRISPR system. Elife 3:e03703. https://doi.org/10.7554/eLife.03703
Ryu S-M, Koo T, Kim K, Lim K, Baek G, Kim S-T, Kim HS, Kim D, Lee H, Chung E, Kim J-S (2018) Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat Biotechnol 36:536
Schwartz CM, Hussain MS, Blenner M, Wheeldon I (2016) Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR−Cas9 mediated genome editing in Yarrowia lipolytica. ACS Synth Biol 5:356–359. https://doi.org/10.1021/acssynbio.5b00162
Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu J-L, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686
Shechner DM, Hacisuleyman E, Younger ST, Rinn JL (2015) Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat Methods 12:664–670. https://doi.org/10.1038/nmeth.3433
Shiraki T, Kawakami K (2018) A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish. Sci Rep 8:13366. https://doi.org/10.1038/s41598-018-31476-5
Song L, Ouedraogo J-P, Kolbusz M, Nguyen TTM, Tsang A (2018) Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger. PLoS ONE 13:e0202868–e0202868. https://doi.org/10.1371/journal.pone.0202868
Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, Xi Y (2015) Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep 5:10342
Svitashev S, Schwartz C, Lenderts B, Young JK, Mark Cigan A (2016) Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun 7:13274. https://doi.org/10.1038/ncomms13274
Tang X, Zheng X, Qi Y, Zhang D, Cheng Y, Tang A, Voytas DF, Zhang Y (2016) A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol Plant 9:1088–1091. https://doi.org/10.1016/j.molp.2016.05.001
Tang W, Hu JH, Liu DR (2017a) Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation. Nat Commun 8:15939. https://doi.org/10.1038/ncomms15939
Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y (2017b) A CRISPR—Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants 3:1–5. https://doi.org/10.1038/nplants.2017.18
Tang X, Ren Q, Yang L, Bao Y, Zhong Z, He Y, Liu S, Qi C, Liu B, Wang Y, Sretenovic S, Zhang Y, Zheng X, Zhang T, Qi Y, Zhang Y (2019) Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol J 17:1431–1445. https://doi.org/10.1111/pbi.13068
Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32:569–576. https://doi.org/10.1038/nbt.2908
Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet 17:29–35. https://doi.org/10.1016/S0168-9525(00)02166-1
Wang S, Shi Z, Liu W, Jules J, Feng X (2006) Development and validation of vectors containing multiple siRNA expression cassettes for maximizing the efficiency of gene silencing. BMC Biotechnol 6:50. https://doi.org/10.1186/1472-6750-6-50
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918. https://doi.org/10.1016/j.cell.2013.04.025
Wang M, Zuris JA, Meng F, Rees H, Sun S, Deng P, Han Y, Gao X, Pouli D, Wu Q, Georgakoudi I, Liu DR, Xu Q (2016) Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc Natl Acad Sci 113:2868–2873. https://doi.org/10.1073/pnas.1520244113
Wang M, Mao Y, Lu Y, Wang Z, Tao X, Zhu J-K (2018) Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems. J Integr Plant Biol 60:626–631. https://doi.org/10.1111/jipb.12667
Woo JW, Kim J, Il KS, Corvalán C, Cho SW, Kim H, Kim S-G, Kim S-T, Choe S, Kim J-S (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164. https://doi.org/10.1038/nbt.3389
Wyman C, Kanaar R (2006) DNA double-strand break repair: all’s well that ends well. Annu Rev Genet 40:363–383. https://doi.org/10.1146/annurev.genet.40.110405.090451
Wyvekens N, Topkar VV, Khayter C, Joung JK, Tsai SQ (2015) Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum Gene Ther 26:425–431. https://doi.org/10.1089/hum.2015.084
Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570–3575. https://doi.org/10.1073/pnas.1420294112
Xing H-L, Dong L, Wang Z-P, Zhang H-Y, Han C-Y, Liu B, Wang X-C, Chen Q-J (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327. https://doi.org/10.1186/s12870-014-0327-y
Xu L, Zhao L, Gao Y, Xu J, Han R (2017a) Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA. Nucleic Acids Res 45:e28–e28. https://doi.org/10.1093/nar/gkw1048
Xu R, Qin R, Li H, Li D, Li L, Wei P, Yang J (2017b) Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol J 15:713–717. https://doi.org/10.1111/pbi.12669
Xu R, Qin R, Li H, Li J, Yang J, Wei P (2018) Enhanced genome editing in rice using single transcript unit CRISPR- Lb Cpf1 systems. Plant Biotechnol J. https://doi.org/10.1111/pbi.13028
Xue Z, Wu M, Wen K, Ren M, Long L, Zhang X, Gao G (2014) CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila. G3 (Bethesda) 4:2167–2173. https://doi.org/10.1534/g3.114.014159
Yan Q, Xu K, Xing J, Zhang T, Wang X, Wei Z, Ren C, Liu Z, Shao S, Zhang Z (2016) Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure. Sci Rep 6:38970. https://doi.org/10.1038/srep38970
Yin H, Song C-Q, Suresh S, Kwan S-Y, Wu Q, Walsh S, Ding J, Bogorad RL, Zhu LJ, Wolfe SA, Koteliansky V, Xue W, Langer R, Anderson DG (2018) Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. Nat Chem Biol 14:311
Yoshioka S, Fujii W, Ogawa T, Sugiura K, Naito K (2015) Development of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells. Sci Rep 5:18341. https://doi.org/10.1038/srep18341
Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La RM, Tsai JC, Weissman JS, Dueber JE, Qi LS (2015) Resource engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160:339–350. https://doi.org/10.1016/j.cell.2014.11.052
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038
Zetsche B, Heidenreich M, Mohanraju P, Kneppers J, Degennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, Van Der OJ, Sciences C, Sciences F, Academy R (2017) Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array. Nat Biotechnol 35:31–34. https://doi.org/10.1038/nbt.3737.Multiplex
Zhang W-W, Matlashewski G (2015) CRISPR-Cas9-mediated genome editing in Leishmania donovani. MBio 6:e00861–e00861. https://doi.org/10.1128/mBio.00861-15
Zhang Z, Mao Y, Ha S, Liu W, Botella JR, Zhu J-K (2016) A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Rep 35:1519–1533. https://doi.org/10.1007/s00299-015-1900-z
Zhang Y, Wang J, Wang Z, Zhang Y, Shi S, Nielsen J, Liu Z (2019) A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10:1053. https://doi.org/10.1038/s41467-019-09005-3
Zheng X, Zheng P, Zhang K, Cairns TC, Meyer V, Sun J, Ma Y (2018) 55S rRNA promoter for guide RNA expression enabled highly efficient CRISPR/Cas9 genome editing in Aspergillus niger. ACS Synth Biol 8:1568–1574
Zhong G, Wang H, Li Y, Tran MH, Farzan M (2017) Cpf1 proteins excise CRISPR RNAs from mRNA transcripts in mammalian cells. Nat Chem Biol 13:839–841. https://doi.org/10.1038/nchembio.2410
Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42:10903–10914. https://doi.org/10.1093/nar/gku806
Acknowledgements
This work was supported by NSF Plant Genome Research Project Grant (1740874) and the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04659 and Accession #1016432.
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Hsieh-Feng, V., Yang, Y. Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing. aBIOTECH 1, 123–134 (2020). https://doi.org/10.1007/s42994-019-00014-w
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DOI: https://doi.org/10.1007/s42994-019-00014-w