- Email: [email protected]
CRISPR-Cpf1: A New Tool for Plant Genome Editing
professor are likely to stay with you for the rest of your professional life. I was taught to be passionate about my work, to follow my dreams, work hard, and never give up. These are the values that I would also like to pass to my students. My advice to them is just become excited about what you do. If you are passionate about your work, you will become the best in it. Follow your dreams and do not let minor setbacks and frustrations stand in your way. When I think about my research career, and myself, two memorable moments stand out. One is getting a PhD, which is one of the most enjoyable events that you will not forget. You will feel a tremendous sense of achievement and pride. For the first time, you will feel that you know as much or more about your PhD research topic than any of your examiners. This is your thesis, your work, and you know it better than anybody else. This should be a stress-free event for you, a time to celebrate. The second big moment is getting a permanent job as an independent scientist, which can be a lot of stress. I would advise you to focus on the small successes, allow yourself to be proud and excited about your accomplishments throughout your PhD, and use this to stay motivated and excited about your PhD. It can be a tough career choice in science, and many people will face short postdoc contracts, moving from place to place. At the end of the day, however, working in academia and research is the best job or career you can have! You appreciate interacting with young bright students throughout your career, and I think it keeps me feeling young. There are so many great things you can achieve in academia.
oxygen delivery to specific cells and tissues, for example, to cancer cells or to hypoxic roots in plants.
What has been your biggest mistake? Partly because I get easily excited about various research questions and partly because scientists have to be, to some extent, opportunistic to survive in the world where research funding is limited and highly competitive, my past research effort was not always as focussed as I would have liked it to be. Was it a mistake? If it was, I am not sure that I know how I could have done it differently. I think that for young scientists who want to start their independent research programs, the potential availability of research funding should be at the top of their priorities when looking for employment.
Trends in Plant Science, July 2017, Vol. 22, No. 7
3. Navarro-RóDenas, A. et al. (2015) Laccaria bicolor Aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides). Plant Cell Environ. 38, 2475–2486 4. Zwiazek, J.J. et al. (2017) Significance of oxygen transport through aquaporins. Sci. Rep. 7, 40411 5. Verkman, A.S. et al. (2008) Aquaporins: new players in cancer biology. J. Mol. Med. 86, 523–529 6. Clarkson, D.T. et al. (2000) Root hydraulic conductance: diurnal aquaporin expression and the effects of nutrient stress. J. Exp. Bot. 51, 61–70 7. Henzler, T. et al. (1999) Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus. Planta 210, 50–60 8. Wan, X.C. and Zwiazek, J.J. (1999) Mecuric chloride effects on root water transport in aspen seedlings. Plant Physiol. 121, 939–946 9. Wan, X.C. et al. (1999) Root water flow and growth of aspen (Populus tremuloides) at low root temperatures. Tree Physiol. 19, 879–884
Spotlight
CRISPR-Cpf1: A New Tool for Plant Genome Editing
Given the importance of aquaporins across all organisms do you believe there is a need for more discourse between Syed Shan-e-Ali Zaidi,1 biological disciplines? 2
Yes, we need to talk, we need to learn, and we need to exchange ideas between biologists representing different disciplines. We already do a decent job of exploiting the era of fast electronic communication and social media that increasing numbers of people use in science. I have always found at least one or two talks at multidisciplinary conferences that inspire me and get me excited about new research ideas. We have a good track record of conferences that offer a broad forum for biological and biomedical researchers to exchange their knowledge of aquaporins and, with the large potential for cross-disciplinary applications of aquaporin research, I would like to see the tradition of these international aquaporin conferences to continue in the What big questions interest you future. We do have a lot to learn from each in the long term? Understanding transcellular oxygen other. transport and the role of aquaporins in http://dx.doi.org/10.1016/j.tplants.2017.05.003 this process. This is a very exciting area of research of broad importance since it References 1. Lehto, T. and Zwiazek, J.J. (2011) Ectomycorrhizas and water relations of trees: a review. Mycorrhiza 21, 71–90 could open possibilities of regulating
550
2. Muhsin, T.M. and Zwiazek, J.J. (2002) Ectomycorrhizas increase apoplastic water transport and root hydraulic conductivity in Ulmus americana seedlings. New Phytol. 153, 153–158
Magdy M. Mahfouz, and Shahid Mansoor1,* Clustered regularly interspaced palindromic repeats (CRISPR)CRISPR-associated proteins (CRISPR-Cas), a groundbreaking genome-engineering tool, has facilitated targeted trait improvement in plants. Recently, CRISPR-CRISPR from Prevotella and Francisella 1 (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free editing in plants, with higher efficiency, specificity, and potentially wider applications than CRISPR-Cas9.
CRISPR-Cas forms part of the adaptive immune system of prokaryotes, and domestication of this system provided a
CRISPR-Cpf1
(A)
T-rich PAM
5’ 3’
CRISPR-Cas9
(B)
TTTN AAAN
G-rich PAM
Cas9
Cpf1 5’
5’
NGG NCC
3’
5’ 3’
3’
sgRNA
Scky ends
Blunt ends
Figure 1. Plant Genome Engineering with Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR from Prevotella and Francisella 1 (Cpf1) Compared with CRISPR-CRISPR-Associated Proteins (Cas9). In a Cpf1-mediated plant genome-editing system (A), a T-rich region (TTTN) acts as protospacer adjacent motif (PAM) and creates double-stranded breaks (DSBs) distal from recognition site, with 50 staggered ends. In a Cas9-mediated genome-editing system (B), a G-rich region (NGG) acts as PAM, creates DSBs proximal to recognition site (three nucleotides away) and produces blunt ends. In both cases, the DSBs are subsequently repaired by two major cellular mechanisms, nonhomologous end joining (NHEJ) and homology-directed repair (HDR).
revolutionary tool for plant genome engineering. Although developed recently, CRISPR-Cas has been established in important model and crop plants, such as rice, wheat, maize, tomato, potato, tobacco, cotton, soybean, and Arabidopsis thaliana. Important traits, such as cold tolerance, drought tolerance, herbicide resistance, yield increase, quality improvement, virus disease resistance, and fungal disease resistance, can be introduced and/or improved in economically important crops. Moreover, CRISPR-based approaches have produced DNA-free and/or nongenetically modified (GM) crops [1], which have modified traits but lack exogenous DNA; these crops have
been approved for commercial production in the USA [2]. To date, most genome editing with the CRISPR system has used Cas9, a type II nuclease from Streptococcus pyogenes. In type II CRISPR-Cas in the prokaryote immune system, RNase III and the Cas9 protein are involved in the processing of the precursor CRISPR RNA (pre-crRNA) in the presence of the trans-acting crRNA (tracrRNA). The Cas9/guide RNA ribonucleoprotein complex also recognizes the target site and makes a site-specific double-stranded break (DSB). By contrast, in the type I and type III systems, the recognition and cleavage of target sites involve several Cas proteins. Given the simplicity and efficiency of the type II system, Cas9
proteins are widely used for genome editing. In the simplified system used for genome editing, the tracrRNA and crRNA are replaced by a single-guide RNA (sgRNA) [3]. Cpf1, a recently introduced class II type V endonuclease, has novel, superior features that SpCas9 lacks. First, and the most important from the perspective of gene editing in plants, Cpf1 generates cohesive ends with four- or five-nucleotide (nt) overhangs, compared with SpCas9, which produces blunt ends (Figure 1). These cohesive DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1cleaved site using complementary DNA
Trends in Plant Science, July 2017, Vol. 22, No. 7
551
ends through a mechanism known as ‘homology-directed repair’ (HDR). This should significantly enhance gene insertion at a precise genome location, a feature highly desirable but currently challenging in plants. Second, Cpf1 cleaves the target DNA molecule with a single crRNA that is shorter than the sgRNA for SpCas9 (43 nt versus 100 nt). Therefore, Cpf1-mediated genome editing with a chemically synthesized crRNA can be achieved at lower cost than editing with SpCas9 and a synthetic sgRNA. Third, Cpf1 recognizes a T-rich protospacer adjacent motif (PAM); it requires 50 -TTTN-30 (or 50 -TTTV-30 ; V = A, C, or G, in some cases) PAM sequences, compared with the G-rich, NGG, PAM sequence in Cas9. In addition, Cpf1 recognizes a PAM that is 50 instead of 30 of the target site (Figure 1). Fourth, Cpf1 contains not only DSBinducing activity, but also RNase III activity for pre-crRNA processing. This activity can be exploited for efficient multiplex genome engineering via tandemly arrayed pre-crRNA-expressing constructs that produce multiple mature crRNAs processed by Cpf1. Finally, one of the major shortcomings of the Cas9 is its high offtarget activity, especially in mammalian cells. Although off-target activities are less of a concern in plants, studies have shown that Cpf1 exhibits little to no offtarget activities in plant cells, consistent with reports in mammalian cells [4,5]. Among several proteins in the Cpf1 family, LbCpf1 from Lachnospiraceae bacterium ND 2006 and AsCpf1 from Acidaminococcus sp. BV3L6 function effectively [6]. Recent studies have shown the efficacy of CRISPR-Cpf1 for efficient and precise genome editing in plants. Endo et al, used Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) compared with known Cpf1 proteins, for targeted mutagenesis in tobacco and rice [7]. Yin et al. knocked out a positive regulator of stomatal development (EPFL9) using both Cas9 and Cpf1 in rice and concluded that, ‘Despite
552
Trends in Plant Science, July 2017, Vol. 22, No. 7
the fact that low gRNA on-target activity was predicted by DESKGEN in the Cpf1 target site (7% probability for the Cpf1 system, vs. 69% for the Cas9 system), more than double the percentage of edited plants were observed with the LbCpf1 system in T0’ [8]. Wang et al. used the Cpf1 system for multiplexed gene editing in rice by editing four OsBEL genes [9], and Xu et al. also reported an efficient and heritable mutagenesis of OsPDS and OsBEL genes in rice plants. Hu et al. and Begemann et al. also reported similar results with CRISPR-Cpf1-mediated targeted mutagenesis in rice [10,11]. Kim et al. described the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco, and showed successful mutations with no observed off-target effects [12]. Further studies will explore the advantages and disadvantages of Cpf1 in plant genome-engineering applications. For example, although Cpf1 enables genome editing with the least off-target activity, its stringent PAM sequence limits the range of target sequences. Most of the Cpf1mediated mutations observed in rice were relatively long deletions, compared with the common short indels [one to two base pairs (bp)] generated by Cas9. Moreover, instead of biallelic mutations usually carried out by Cas9, the mutated rice lines in which Cpf1 was used were either chimeric or heterozygous [5]. This suggests that Cpf1-induced mutants are largely somatic and are produced as a result of non-homologous end-joining repair on the 4–5-nt 50 overhangs resulting from the staggered cutting of Cpf1 (Figure 1). However, further work should lead to plausable answers based on experimental evidence. The expression of Cpf1 in plants can be enhanced with codon optimization and the addition of a nuclear localization signal, translational enhancer, transcriptional terminator, and strong constitutive promoters; it is likely that a combination of a stable expression
system and these additional tweaks will contribute to improving the genome-editing activity of Cpf1 in plant cells. In addition to Cpf1, at least 53 other class II CRISPR-Cas candidates have been characterized. Among them, C2c2 nucleases have the unique property of targeting single-stranded RNA. This offers the possibility of enabling gene-knockdown applications by targeting mRNAs. In addition, C2c2 also serves dual nuclease activity, similar to Cpf1, although the application of C2c2 in eukaryotes, including plants, remains to be explored. Applications of genome editing in plants can broadly expand using CRISPR-Cpf1, facilitating approaches such as genomewide functional screening based on gene knockouts, transcriptional repression using catalytically inactivated Cpf1 (dCpf1), or transcriptional activation using dCpf1 fused with a transcription activator domain, analysis of chromatin dynamics using dCpf1 fused with fluorescent proteins, epigenome editing with dCpf1 fused to epigenetic modifiers, and the tracking of cell lineages with DNA-barcoding techniques. These advances expand the molecular toolbox of plant genome engineering and the possibilities for the targeted improvement of crop traits, such as yield, quality enhancement of produce, and input use efficiency, for ensuring sustainable food security. 1
National Institute for Biotechnology and Genetic
Engineering (NIBGE), Faisalabad, Pakistan 2 Laboratory for Genome Engineering, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Thuwal, Saudi Arabia *Correspondence: [email protected] (S. Mansoor). http://dx.doi.org/10.1016/j.tplants.2017.05.001 References 1. Kanchiswamy, C.N. et al. (2015) Non-GMO genetically edited crop plants. Trends Biotechnol. 33, 489–491 2. Waltz, E. (2016) CRISPR-edited crops free to enter market, skip regulation. Nat. Biotechnol. 34, 582 3. Jinek, M. et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 4. Tang, X. et al. (2017) A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17018
5. Xu, R. et al. (2017) Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol. J. 15, 713–717
external environment signals. Root hairs are single plant cells that can expand to several hundred-fold their original size, and have emerged as an excellent model 7. Endo, A. et al. (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella nov- system for studying cell size regulation. icida. Sci. Rep. 6, 38169 Root hair development varies by plant 8. Yin, X. et al. (2017) CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene species; it occurs either randomly, starting EPFL9 in rice. Plant Cell Rep. 36, 745–757 with an asymmetrical cell division, or via a 9. Wang, M. et al. (2017) Multiplex gene editing in rice using position-dependent mechanism. The latter the CRISPR-Cpf1 system. Mol. Plant Published online March 16, 2017. http://dx.doi.org/10.1016/j. mechanism is better studied and occurs in molp.2017.03.001 the model plant Arabidopsis thaliana, 10. Begemann, M.B. et al. (2016) Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucle- where root hair cells, or trichoblasts, and ases. bioRxiv Published online February 20, 2017. http:// nonhair cells, or atrichoblasts, differentiate dx.doi.org/10.1101/109983 from the epidermal cell layer. A well11. Hu, X. et al. (2017) Targeted mutagenesis in rice using defined developmental program and CRISPR-Cpf1 system. J. Genet. Genomics 44, 71–73 12. Kim, H. et al. (2017) CRISPR/Cpf1-mediated DNA-free multiple environmental signals coupled to plant genome editing. Nat. Commun. 8, 14406 several hormones are integrated to define the final size of root hairs (Figure 1). Root hair size has vital physiological implications Spotlight for the plant, determining the surface area: volume ratio of the all the roots exposed to the nutrient pools, thereby likely impacting nutrient uptake rates. Although the final hair size is of fundamental importance, the molecular mechanisms that control it remained largely unknown until recently. The developmental program, hormones, and environmental cues all converge to Eliana Marzol,1,y regulate the expression of the single basic Cecilia Borassi,1,y 1,y helix-loop-helix (bHLH) transcription factor Silvina Paola Denita Juárez, (TF) RSL4, which controls polar growth. Silvina Mangano,1 and Previous studies have discussed these 1, José M. Estevez * three individual factors in detail [1–3]. Here, we discuss recent progress toward the Root hair growth dramatically elucidation of how the final size of root hair expands the root surface area, cells is fine-tuned by the master regulator, thus facilitating water and nutrient RSL4. 6. Kleinstiver, B.P. et al. (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874
RSL4 Takes Control: Multiple Signals, One Transcription Factor
uptake. Until recently, the molecular mechanism underlying root hair growth was unknown. Recent studies have revealed that the transcription factor ROOT HAIR DEFECTIVE 6 LIKE 4 (RSL4) coordinates hormonal, environmental, and developmental factors to trigger polar growth. One of the most intriguing questions in modern biology is how cells regulate their size. The rate at which cells grow is determined by both cell intrinsic factors and
Specification of epidermal cell differentiation is a highly regulated process. A TF complex comprising WEREWOLF (WER), GLABRA 3 (GL3)/ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1, induces the expression of GLABRA 2 (GL2), which inhibits root hair cell fate, suppressing ROOT HAIR DEFECTIVE 6 (RHD6) [4]. In trichoblasts, the lack of GL2 allows the expression of RDH6, leading to root hair initiation, a process that is controlled by the interplay between several genes, such as RDH6–RSL1 [5]. Recently, it was
shown that expression of RSL4 under the control of the GL2 promoter, in both the wild-type and hairless rhd6 mutant backgrounds, induced root hair growth in atrichoblasts. This suggests that RSL4 expression induces root hair formation and growth independently of RHD6– RSL1 [5]. This is likely because RSL4 regulates several genes involved in different key cell processes required for root hair growth (Figure 1). Together with the developmental and genetic pathway, hormones, such as auxin (IAA for indole-3-acetic acid), ethylene (Et), cytokinin (CK), and strigolactones (SLs) are important triggers of root hair cell growth in trichoblasts [1–3]. Mechanistically, auxin needs to be sensed in situ in the root hair cells to trigger cell expansion (Figure 1). The slow transcriptional IAA response involves members of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) family and their co-receptor AUXIN/INDOLE 3-ACETIC ACID (Aux/ IAA), and the concomitant release of AUXIN RESPONSE FACTORS (ARFs). ARFs bind to cis-auxin response elements (Aux-REs) in the promoters of early IAA response genes to trigger downstream responses [2]. Recently, it was shown that several ARFs directly upregulate RSL4 expression several-fold, linking IAA stimulation to RSL4 expression at the molecular level [6,7]. In addition, RSL4 was shown to promote reactive oxygen species (ROS) production by regulating the expression of two NADPH oxidases, C and J [also known as RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins] and several Class III apoplastic peroxidases (PERs) [5,7]. Chemical or genetic interference with ROS balance or peroxidase activity affects the final size of root hair cells. IAA stimulation of ROS production in root hair cells requires not only RSL4, but also RSL2, but how this hormonal program is coordinated remains to be determined [7]. Overall, these findings established a molecular link between IAA-regulated ARFs–RSL4
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oxygen delivery to specific cells and tissues, for example, to cancer cells or to hypoxic roots in plants.
What has been your biggest mistake? Partly because I get easily excited about various research questions and partly because scientists have to be, to some extent, opportunistic to survive in the world where research funding is limited and highly competitive, my past research effort was not always as focussed as I would have liked it to be. Was it a mistake? If it was, I am not sure that I know how I could have done it differently. I think that for young scientists who want to start their independent research programs, the potential availability of research funding should be at the top of their priorities when looking for employment.
Trends in Plant Science, July 2017, Vol. 22, No. 7
3. Navarro-RóDenas, A. et al. (2015) Laccaria bicolor Aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides). Plant Cell Environ. 38, 2475–2486 4. Zwiazek, J.J. et al. (2017) Significance of oxygen transport through aquaporins. Sci. Rep. 7, 40411 5. Verkman, A.S. et al. (2008) Aquaporins: new players in cancer biology. J. Mol. Med. 86, 523–529 6. Clarkson, D.T. et al. (2000) Root hydraulic conductance: diurnal aquaporin expression and the effects of nutrient stress. J. Exp. Bot. 51, 61–70 7. Henzler, T. et al. (1999) Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus. Planta 210, 50–60 8. Wan, X.C. and Zwiazek, J.J. (1999) Mecuric chloride effects on root water transport in aspen seedlings. Plant Physiol. 121, 939–946 9. Wan, X.C. et al. (1999) Root water flow and growth of aspen (Populus tremuloides) at low root temperatures. Tree Physiol. 19, 879–884
Spotlight
CRISPR-Cpf1: A New Tool for Plant Genome Editing
Given the importance of aquaporins across all organisms do you believe there is a need for more discourse between Syed Shan-e-Ali Zaidi,1 biological disciplines? 2
Yes, we need to talk, we need to learn, and we need to exchange ideas between biologists representing different disciplines. We already do a decent job of exploiting the era of fast electronic communication and social media that increasing numbers of people use in science. I have always found at least one or two talks at multidisciplinary conferences that inspire me and get me excited about new research ideas. We have a good track record of conferences that offer a broad forum for biological and biomedical researchers to exchange their knowledge of aquaporins and, with the large potential for cross-disciplinary applications of aquaporin research, I would like to see the tradition of these international aquaporin conferences to continue in the What big questions interest you future. We do have a lot to learn from each in the long term? Understanding transcellular oxygen other. transport and the role of aquaporins in http://dx.doi.org/10.1016/j.tplants.2017.05.003 this process. This is a very exciting area of research of broad importance since it References 1. Lehto, T. and Zwiazek, J.J. (2011) Ectomycorrhizas and water relations of trees: a review. Mycorrhiza 21, 71–90 could open possibilities of regulating
550
2. Muhsin, T.M. and Zwiazek, J.J. (2002) Ectomycorrhizas increase apoplastic water transport and root hydraulic conductivity in Ulmus americana seedlings. New Phytol. 153, 153–158
Magdy M. Mahfouz, and Shahid Mansoor1,* Clustered regularly interspaced palindromic repeats (CRISPR)CRISPR-associated proteins (CRISPR-Cas), a groundbreaking genome-engineering tool, has facilitated targeted trait improvement in plants. Recently, CRISPR-CRISPR from Prevotella and Francisella 1 (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free editing in plants, with higher efficiency, specificity, and potentially wider applications than CRISPR-Cas9.
CRISPR-Cas forms part of the adaptive immune system of prokaryotes, and domestication of this system provided a
CRISPR-Cpf1
(A)
T-rich PAM
5’ 3’
CRISPR-Cas9
(B)
TTTN AAAN
G-rich PAM
Cas9
Cpf1 5’
5’
NGG NCC
3’
5’ 3’
3’
sgRNA
Scky ends
Blunt ends
Figure 1. Plant Genome Engineering with Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR from Prevotella and Francisella 1 (Cpf1) Compared with CRISPR-CRISPR-Associated Proteins (Cas9). In a Cpf1-mediated plant genome-editing system (A), a T-rich region (TTTN) acts as protospacer adjacent motif (PAM) and creates double-stranded breaks (DSBs) distal from recognition site, with 50 staggered ends. In a Cas9-mediated genome-editing system (B), a G-rich region (NGG) acts as PAM, creates DSBs proximal to recognition site (three nucleotides away) and produces blunt ends. In both cases, the DSBs are subsequently repaired by two major cellular mechanisms, nonhomologous end joining (NHEJ) and homology-directed repair (HDR).
revolutionary tool for plant genome engineering. Although developed recently, CRISPR-Cas has been established in important model and crop plants, such as rice, wheat, maize, tomato, potato, tobacco, cotton, soybean, and Arabidopsis thaliana. Important traits, such as cold tolerance, drought tolerance, herbicide resistance, yield increase, quality improvement, virus disease resistance, and fungal disease resistance, can be introduced and/or improved in economically important crops. Moreover, CRISPR-based approaches have produced DNA-free and/or nongenetically modified (GM) crops [1], which have modified traits but lack exogenous DNA; these crops have
been approved for commercial production in the USA [2]. To date, most genome editing with the CRISPR system has used Cas9, a type II nuclease from Streptococcus pyogenes. In type II CRISPR-Cas in the prokaryote immune system, RNase III and the Cas9 protein are involved in the processing of the precursor CRISPR RNA (pre-crRNA) in the presence of the trans-acting crRNA (tracrRNA). The Cas9/guide RNA ribonucleoprotein complex also recognizes the target site and makes a site-specific double-stranded break (DSB). By contrast, in the type I and type III systems, the recognition and cleavage of target sites involve several Cas proteins. Given the simplicity and efficiency of the type II system, Cas9
proteins are widely used for genome editing. In the simplified system used for genome editing, the tracrRNA and crRNA are replaced by a single-guide RNA (sgRNA) [3]. Cpf1, a recently introduced class II type V endonuclease, has novel, superior features that SpCas9 lacks. First, and the most important from the perspective of gene editing in plants, Cpf1 generates cohesive ends with four- or five-nucleotide (nt) overhangs, compared with SpCas9, which produces blunt ends (Figure 1). These cohesive DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1cleaved site using complementary DNA
Trends in Plant Science, July 2017, Vol. 22, No. 7
551
ends through a mechanism known as ‘homology-directed repair’ (HDR). This should significantly enhance gene insertion at a precise genome location, a feature highly desirable but currently challenging in plants. Second, Cpf1 cleaves the target DNA molecule with a single crRNA that is shorter than the sgRNA for SpCas9 (43 nt versus 100 nt). Therefore, Cpf1-mediated genome editing with a chemically synthesized crRNA can be achieved at lower cost than editing with SpCas9 and a synthetic sgRNA. Third, Cpf1 recognizes a T-rich protospacer adjacent motif (PAM); it requires 50 -TTTN-30 (or 50 -TTTV-30 ; V = A, C, or G, in some cases) PAM sequences, compared with the G-rich, NGG, PAM sequence in Cas9. In addition, Cpf1 recognizes a PAM that is 50 instead of 30 of the target site (Figure 1). Fourth, Cpf1 contains not only DSBinducing activity, but also RNase III activity for pre-crRNA processing. This activity can be exploited for efficient multiplex genome engineering via tandemly arrayed pre-crRNA-expressing constructs that produce multiple mature crRNAs processed by Cpf1. Finally, one of the major shortcomings of the Cas9 is its high offtarget activity, especially in mammalian cells. Although off-target activities are less of a concern in plants, studies have shown that Cpf1 exhibits little to no offtarget activities in plant cells, consistent with reports in mammalian cells [4,5]. Among several proteins in the Cpf1 family, LbCpf1 from Lachnospiraceae bacterium ND 2006 and AsCpf1 from Acidaminococcus sp. BV3L6 function effectively [6]. Recent studies have shown the efficacy of CRISPR-Cpf1 for efficient and precise genome editing in plants. Endo et al, used Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) compared with known Cpf1 proteins, for targeted mutagenesis in tobacco and rice [7]. Yin et al. knocked out a positive regulator of stomatal development (EPFL9) using both Cas9 and Cpf1 in rice and concluded that, ‘Despite
552
Trends in Plant Science, July 2017, Vol. 22, No. 7
the fact that low gRNA on-target activity was predicted by DESKGEN in the Cpf1 target site (7% probability for the Cpf1 system, vs. 69% for the Cas9 system), more than double the percentage of edited plants were observed with the LbCpf1 system in T0’ [8]. Wang et al. used the Cpf1 system for multiplexed gene editing in rice by editing four OsBEL genes [9], and Xu et al. also reported an efficient and heritable mutagenesis of OsPDS and OsBEL genes in rice plants. Hu et al. and Begemann et al. also reported similar results with CRISPR-Cpf1-mediated targeted mutagenesis in rice [10,11]. Kim et al. described the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco, and showed successful mutations with no observed off-target effects [12]. Further studies will explore the advantages and disadvantages of Cpf1 in plant genome-engineering applications. For example, although Cpf1 enables genome editing with the least off-target activity, its stringent PAM sequence limits the range of target sequences. Most of the Cpf1mediated mutations observed in rice were relatively long deletions, compared with the common short indels [one to two base pairs (bp)] generated by Cas9. Moreover, instead of biallelic mutations usually carried out by Cas9, the mutated rice lines in which Cpf1 was used were either chimeric or heterozygous [5]. This suggests that Cpf1-induced mutants are largely somatic and are produced as a result of non-homologous end-joining repair on the 4–5-nt 50 overhangs resulting from the staggered cutting of Cpf1 (Figure 1). However, further work should lead to plausable answers based on experimental evidence. The expression of Cpf1 in plants can be enhanced with codon optimization and the addition of a nuclear localization signal, translational enhancer, transcriptional terminator, and strong constitutive promoters; it is likely that a combination of a stable expression
system and these additional tweaks will contribute to improving the genome-editing activity of Cpf1 in plant cells. In addition to Cpf1, at least 53 other class II CRISPR-Cas candidates have been characterized. Among them, C2c2 nucleases have the unique property of targeting single-stranded RNA. This offers the possibility of enabling gene-knockdown applications by targeting mRNAs. In addition, C2c2 also serves dual nuclease activity, similar to Cpf1, although the application of C2c2 in eukaryotes, including plants, remains to be explored. Applications of genome editing in plants can broadly expand using CRISPR-Cpf1, facilitating approaches such as genomewide functional screening based on gene knockouts, transcriptional repression using catalytically inactivated Cpf1 (dCpf1), or transcriptional activation using dCpf1 fused with a transcription activator domain, analysis of chromatin dynamics using dCpf1 fused with fluorescent proteins, epigenome editing with dCpf1 fused to epigenetic modifiers, and the tracking of cell lineages with DNA-barcoding techniques. These advances expand the molecular toolbox of plant genome engineering and the possibilities for the targeted improvement of crop traits, such as yield, quality enhancement of produce, and input use efficiency, for ensuring sustainable food security. 1
National Institute for Biotechnology and Genetic
Engineering (NIBGE), Faisalabad, Pakistan 2 Laboratory for Genome Engineering, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Thuwal, Saudi Arabia *Correspondence: [email protected] (S. Mansoor). http://dx.doi.org/10.1016/j.tplants.2017.05.001 References 1. Kanchiswamy, C.N. et al. (2015) Non-GMO genetically edited crop plants. Trends Biotechnol. 33, 489–491 2. Waltz, E. (2016) CRISPR-edited crops free to enter market, skip regulation. Nat. Biotechnol. 34, 582 3. Jinek, M. et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 4. Tang, X. et al. (2017) A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17018
5. Xu, R. et al. (2017) Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol. J. 15, 713–717
external environment signals. Root hairs are single plant cells that can expand to several hundred-fold their original size, and have emerged as an excellent model 7. Endo, A. et al. (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella nov- system for studying cell size regulation. icida. Sci. Rep. 6, 38169 Root hair development varies by plant 8. Yin, X. et al. (2017) CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene species; it occurs either randomly, starting EPFL9 in rice. Plant Cell Rep. 36, 745–757 with an asymmetrical cell division, or via a 9. Wang, M. et al. (2017) Multiplex gene editing in rice using position-dependent mechanism. The latter the CRISPR-Cpf1 system. Mol. Plant Published online March 16, 2017. http://dx.doi.org/10.1016/j. mechanism is better studied and occurs in molp.2017.03.001 the model plant Arabidopsis thaliana, 10. Begemann, M.B. et al. (2016) Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucle- where root hair cells, or trichoblasts, and ases. bioRxiv Published online February 20, 2017. http:// nonhair cells, or atrichoblasts, differentiate dx.doi.org/10.1101/109983 from the epidermal cell layer. A well11. Hu, X. et al. (2017) Targeted mutagenesis in rice using defined developmental program and CRISPR-Cpf1 system. J. Genet. Genomics 44, 71–73 12. Kim, H. et al. (2017) CRISPR/Cpf1-mediated DNA-free multiple environmental signals coupled to plant genome editing. Nat. Commun. 8, 14406 several hormones are integrated to define the final size of root hairs (Figure 1). Root hair size has vital physiological implications Spotlight for the plant, determining the surface area: volume ratio of the all the roots exposed to the nutrient pools, thereby likely impacting nutrient uptake rates. Although the final hair size is of fundamental importance, the molecular mechanisms that control it remained largely unknown until recently. The developmental program, hormones, and environmental cues all converge to Eliana Marzol,1,y regulate the expression of the single basic Cecilia Borassi,1,y 1,y helix-loop-helix (bHLH) transcription factor Silvina Paola Denita Juárez, (TF) RSL4, which controls polar growth. Silvina Mangano,1 and Previous studies have discussed these 1, José M. Estevez * three individual factors in detail [1–3]. Here, we discuss recent progress toward the Root hair growth dramatically elucidation of how the final size of root hair expands the root surface area, cells is fine-tuned by the master regulator, thus facilitating water and nutrient RSL4. 6. Kleinstiver, B.P. et al. (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874
RSL4 Takes Control: Multiple Signals, One Transcription Factor
uptake. Until recently, the molecular mechanism underlying root hair growth was unknown. Recent studies have revealed that the transcription factor ROOT HAIR DEFECTIVE 6 LIKE 4 (RSL4) coordinates hormonal, environmental, and developmental factors to trigger polar growth. One of the most intriguing questions in modern biology is how cells regulate their size. The rate at which cells grow is determined by both cell intrinsic factors and
Specification of epidermal cell differentiation is a highly regulated process. A TF complex comprising WEREWOLF (WER), GLABRA 3 (GL3)/ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1, induces the expression of GLABRA 2 (GL2), which inhibits root hair cell fate, suppressing ROOT HAIR DEFECTIVE 6 (RHD6) [4]. In trichoblasts, the lack of GL2 allows the expression of RDH6, leading to root hair initiation, a process that is controlled by the interplay between several genes, such as RDH6–RSL1 [5]. Recently, it was
shown that expression of RSL4 under the control of the GL2 promoter, in both the wild-type and hairless rhd6 mutant backgrounds, induced root hair growth in atrichoblasts. This suggests that RSL4 expression induces root hair formation and growth independently of RHD6– RSL1 [5]. This is likely because RSL4 regulates several genes involved in different key cell processes required for root hair growth (Figure 1). Together with the developmental and genetic pathway, hormones, such as auxin (IAA for indole-3-acetic acid), ethylene (Et), cytokinin (CK), and strigolactones (SLs) are important triggers of root hair cell growth in trichoblasts [1–3]. Mechanistically, auxin needs to be sensed in situ in the root hair cells to trigger cell expansion (Figure 1). The slow transcriptional IAA response involves members of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) family and their co-receptor AUXIN/INDOLE 3-ACETIC ACID (Aux/ IAA), and the concomitant release of AUXIN RESPONSE FACTORS (ARFs). ARFs bind to cis-auxin response elements (Aux-REs) in the promoters of early IAA response genes to trigger downstream responses [2]. Recently, it was shown that several ARFs directly upregulate RSL4 expression several-fold, linking IAA stimulation to RSL4 expression at the molecular level [6,7]. In addition, RSL4 was shown to promote reactive oxygen species (ROS) production by regulating the expression of two NADPH oxidases, C and J [also known as RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins] and several Class III apoplastic peroxidases (PERs) [5,7]. Chemical or genetic interference with ROS balance or peroxidase activity affects the final size of root hair cells. IAA stimulation of ROS production in root hair cells requires not only RSL4, but also RSL2, but how this hormonal program is coordinated remains to be determined [7]. Overall, these findings established a molecular link between IAA-regulated ARFs–RSL4
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