Construct design for CRISPR/Cas-based genome editing in plants

Many Cas nucleases (e.g., SpCas9-NRRH, SpG, SpCas9-NG) that can target non-canonical protospacer adjacent motifs (PAMs) have been developed for plant genome editing.Near-PAMless Cas nuclease SpRY has been optimized for plant genome editing to increase the flexibility of gRNA design.A next-generation genome editing technology, prime editing, has been tested in many plants, including Arabidopsis, rice, maize, potato, and tomato.Multiplex clustered regularly interspaced short palindromic repeat (CRISPR) systems based on tRNA/gRNA or Csy4 work better for Cas9 and a hammerhead and hepatitis delta virus (HH-HDV)-based system works better for Cas12a.A multiplex CRISPR system expressing up to 24 gRNAs has been tested in plants.Use of multiple introns in the Cas gene dramatically improves editing efficacy.Improved pegRNA design significantly improves the efficiency of the prime editor. CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing. CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing. Genome editing can be defined as a targeted intervention of genetic materials (i.e., DNA or RNA) in living organisms to deliberately alter their sequences. Although genome editing can target both DNA and RNA, here we only review DNA editing. DNA editing mainly relies on the introduction of in vivo DNA double-stranded breaks (DSBs) induced by the engineered sequence-specific nucleases (SSNs) programmed to recognize predefined sites in a genome. The induced DSBs are then repaired by cellular DNA repair mechanisms, namely non-homologous end-joining (NHEJ) and homology-directed repair (HDR) (Figure 1). The repair of DSBs by NHEJ results in mutation at the break site, largely via imprecise sequence insertions or deletions (indels), disrupting the native structure and function of the targeted sequences (e.g., genes, promoters). In addition, NHEJ can mediate targeted sequence insertion or replacement when a suitable DNA fragment is provided [1.Lu Y. et al.Targeted, efficient sequence insertion and replacement in rice.Nat. Biotechnol. 2020; 38: 1402-1407Crossref PubMed Scopus (28) Google Scholar]. By contrast, repair by HDR can precisely introduce predefined sequences carried by a donor DNA template (Figure 1). The SSNs, with the capacity to introduce DSB in DNA, are referred to as the key elements in genome editing technologies and include meganucleases [2.Bogdanove A.J. et al.Engineering altered protein-DNA recognition specificity.Nucleic Acids Res. 2018; 46: 4845-4871Crossref PubMed Scopus (14) Google Scholar], zinc finger nucleases (ZFNs) [3.Bibikova M. et al.Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.Genetics. 2002; 161: 1169-1175Crossref PubMed Google Scholar], transcription activator-like effector nucleases (TALENs) [4.Christian M. et al.Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1163) Google Scholar], and clustered regularly interspaced short palindromic repeat (CRISPR) systems [5.Zetsche B. et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Google Scholar, 6.Mali P. et al.RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (5536) Google Scholar, 7.Cong L. et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (8358) Google Scholar, 8.Jinek M. et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7291) Google Scholar]. Unlike ZFNs and TALENs, which rely on protein–DNA interaction to define target specificity, CRISPR systems use RNA–DNA interaction to guide the DNA targeting and cleavage, making it a simple, efficient, and inexpensive technology for genetic manipulation. CRISPR systems have now become the leading genome editing technology and have been applied in a wide variety of plant species. Efficient genome editing has been achieved in many dicot and monocot species using diverse CRISPR-Cas systems for fundamental research and crop improvement and the application of CRISPR-Cas technology in plants has been increased dramatically over the past few years [9.Huang T.-K. Puchta H. Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering.Transgenic Res. 2021; (Published online March 1, 2021. https://doi.org/10.1007/s11248-021-00238-x)Crossref Scopus (2) Google Scholar, 10.Zhu H. et al.Applications of CRISPR-Cas in agriculture and plant biotechnology.Nat. Rev. Mol. Cell Biol. 2020; 21: 661-677Crossref PubMed Scopus (80) Google Scholar, 11.Haque E. et al.Application of CRISPR/Cas9 genome editing technology for the improvement of crops cultivated in tropical climates: recent progress, prospects, and challenges.Front. Plant Sci. 2018; 9: 617Crossref PubMed Scopus (44) Google Scholar, 12.Jaganathan D. et al.CRISPR for crop improvement: an update review.Front. Plant Sci. 2018; 9: 985Crossref PubMed Scopus (147) Google Scholar]. Three classes of CRISPR technology are currently available for editing plant genomes [10.Zhu H. et al.Applications of CRISPR-Cas in agriculture and plant biotechnology.Nat. Rev. Mol. Cell Biol. 2020; 21: 661-677Crossref PubMed Scopus (80) Google Scholar,13.Gao C. Genome engineering for crop improvement and future agriculture.Cell. 2021; 184: 1621-1635Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar]. These are CRISPR-Cas nucleases, base editors, and prime editors. CRISPR-Cas nucleases require inducing DSB, whereas base editors and primer editors do not require DSB to edit genomes. Over the past few years, there has been tremendous progress in the development of CRISPR-based technologies. The rapid discovery and development of diverse CRISPR toolboxes thus can make the prospect of selecting a tool for desired application daunting, particularly for researchers new to the genome editing technology. Besides selection of the right CRISPR tools, delivery of CRISPR reagents to plant cells is challenging. In some systems such as mammalian cells, purified protein or mRNA of a Cas protein, as well as the gRNA (see Glossary), can be simultaneously delivered to a zygotic cell. In this way, targeting possibility can be improved by controlling the dosage of Cas proteins and gRNAs. This approach has also been shown to work in plants, but there are still some significant challenges to be overcome. Thus, most frequently, CRISPR reagents are delivered into plants via a construct harboring a Cas gene and at least one gRNA along with the components required for their expression (e.g., promoter, terminator) through Agrobacterium-mediated transformation or particle bombardment. Hence, construct design is a critical step to conduct the CRISPR experiment. Different elements of a CRISPR construct can significantly influence the editing outcome and optimization of Cas gene and gRNA expression are often required to achieve efficient editing [14.Johnson R.A. et al.Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.Plant Mol. 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Here, we review different CRISPR-based genome editing technologies and their technical aspects with the aim to guide users in selecting the appropriate editing technologies and optimizing construct design for various applications. We restrict our discussion to the targeted editing of DNA sequence and refer readers to excellent reviews for other CRISPR applications in plants such as transcriptional regulation [20.Pan C. et al.CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants.Curr. Opin. Plant Biol. 2021; 60: 101980Crossref PubMed Scopus (2) Google Scholar,21.Moradpour M. Abdulah S.N.A. CRISPR/dCas9 platforms in plants: strategies and applications beyond genome editing.Plant Biotechnol. J. 2020; 18: 32-44Crossref PubMed Scopus (29) Google Scholar] and epigenetic editing [22.Miglani G.S. et al.Plant gene expression control using genome- and epigenome-editing technologies.J. Crop Improv. 2020; 34: 1-63Crossref Scopus (1) Google Scholar]. In this section, we discuss different CRISPR reagents and recent developments that progressively increased the applicability and effectiveness of genome editing technologies in plants. This will help identify and select the appropriate technologies and Cas proteins for different applications. Cas9 is currently the most widely used nuclease in CRISPR studies, particularly one isolated from Streptococcus pyogenes (SpCas9). It complexes with a single guide RNA (sgRNA) for DNA targeting and requires a short stretch of nucleotides known as protospacer adjacent motif (PAM) downstream of its target sequence for DNA recognition (Figure 1A). The PAM sequence for SpCas9 is 5′-NGG-3′ (N = A, T, C, G). Once Cas9 recognizes its PAM sequence, the Cas9-sgRNA complex binds to the target sequence and generates a DSB at the target site (Figure 1D). DNA cleavage activity of Cas9 is achieved by the combined effort of two parts of the protein called the recognition domain and the nuclease domains (RuvC and HNH). The recognition domain senses the complementary DNA sequence and the nuclease domains cleave the DNA [23.Jiang F. Doudna J.A. CRISPR-Cas9 structures and mechanisms.Annu. Rev. Biophys. 2017; 46: 505-529Crossref PubMed Scopus (503) Google Scholar]. Despite the widespread use and proven efficacy of SpCas9 for genome editing purpose across a wide range of organisms, it does have certain limitations. Firstly, it often recognizes DNA sequences that share high sequence identity with the target site, resulting in off-target editing. Secondly, the stringent NGG PAM requirement limits the target DNA that can be manipulated with SpCas9. Thirdly, delivery of SpCas9 into plant cell via a viral-based vector is difficult due to its relatively large size that exceeds the cargo capacity of the virus-based vector. To overcome these limitations, several natural and engineered variants of SpCas9 have been developed that recognize alternative PAMs (Table 1). Among them, Staphylococcus aureus Cas9 (SaCa9) is a natural variant and notable one [24.Ran F.A. et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature. 2015; 520: 186-191Crossref PubMed Scopus (1431) Google Scholar]. It recognizes 5′-NNGRRT and its coding sequence is ~1.0 kb shorter than that of SpCas9, thus being suitable to use with virus-based vectors [25.Kaya H. et al.Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9.Sci. Rep. 2016; 6: 26871Crossref PubMed Scopus (62) Google Scholar,26.Steinert J. et al.Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus.Plant J. 2015; 84: 1295-1305Crossref PubMed Google Scholar]. Many engineered SpCas9 variants have also been applied in plant genome editing, including Cas9-NG and xCas9 [27.Ge Z. et al.Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants.Plant Biotechnol. J. 2019; 17: 1865-1867Crossref PubMed Scopus (27) Google Scholar, 28.Hua K. et al.Genome engineering in rice using Cas9 variants that recognize NG PAM sequences.Mol. Plant. 2019; 12: 1003-1014Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29.Li J. et al.Plant genome editing using xCas9 with expanded PAM compatibility.J. Genet. Genomics. 2019; 46: 277-280Crossref PubMed Scopus (12) Google Scholar, 30.Negishi K. et al.An adenine base editor with expanded targeting scope using SpCas9-NGv1 in rice.Plant Biotechnol. 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Plants. 2019; 5: 14-17Crossref PubMed Scopus (72) Google Scholar], as well as iSpyMacCas9 [36.Sretenovic S. et al.Expanding plant genome editing scope by an engineered iSpyMacCas9 system targeting the A-rich PAM sequences.Plant Commun. 2020; 2: 100101Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar]. A remarkable engineered variant of SpCas9 is SpRY, which is capable of targeting almost all PAM sites (NRN>NYN) [37.Walton R.T. et al.Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.Science. 2020; 368: 290-296Crossref PubMed Scopus (189) Google Scholar], and has also been applied in plant genome editing [38.Ren Q. et al.PAM-less plant genome editing using a CRISPR-SpRY toolbox.Nat. Plants. 2021; 7: 25-33Crossref PubMed Scopus (0) Google Scholar,39.Xu Z. et al.SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition.Genome Biol. 2021; 22: 6Crossref PubMed Scopus (19) Google Scholar]. A high-fidelity variant of SpCas9 that has low off-target activity has also been developed (Table 1). Off-target issues can also be reduced by using paired Cas9 nickase [40.Schiml S. et al.The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny.Plant J. 2014; 80: 1139-1150Crossref PubMed Scopus (211) Google Scholar]. Recently, a number of Cas9 variants that recognize non-canonical PAM (e.g., SpCas9-NRRH) have also been applied in plants [41.Li J. et al.Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.Mol. Plant. 2021; 14: 352-360Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar].Table 1CRISPR-Cas nucleases used in plant genome editingCas nucleasePAMMutationKey featuresRefsSpCas9NGGWTHighly efficient[10.Zhu H. et al.Applications of CRISPR-Cas in agriculture and plant biotechnology.Nat. Rev. Mol. Cell Biol. 2020; 21: 661-677Crossref PubMed Scopus (80) Google Scholar,143.Zhang Y. et al.The emerging and uncultivated potential of CRISPR technology in plant science.Nat. Plants. 2019; 5: 778-794Crossref PubMed Scopus (113) Google Scholar]SpCas9-VQRNGAD1135V/R1335Q/T1337RAlternate PAM[19.Yamamoto A. et al.Developing heritable mutations in Arabidopsis thaliana using a modified CRISPR/Cas9 toolkit comprising PAM-altered Cas9 variants and gRNAs.Plant Cell Physiol. 2019; 60: 2255-2262Crossref PubMed Scopus (10) Google Scholar,108.Hu X. et al.Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice.Plant Biotechnol. J. 2018; 16: 292-297Crossref PubMed Scopus (42) Google Scholar,144.Hu X. et al.Expanding the range of CRISPR/Cas9 genome editing in rice.Mol. Plant. 2016; 9: 943-945Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]SpCas9-EQRNGAGD1135E/R1335Q/T1337RAlternate PAM[19.Yamamoto A. et al.Developing heritable mutations in Arabidopsis thaliana using a modified CRISPR/Cas9 toolkit comprising PAM-altered Cas9 variants and gRNAs.Plant Cell Physiol. 2019; 60: 2255-2262Crossref PubMed Scopus (10) Google Scholar]SpCas9-VRERNGCGD1135V/G1218R/R1335E/T1337RAlternate PAM[144.Hu X. et al.Expanding the range of CRISPR/Cas9 genome editing in rice.Mol. Plant. 2016; 9: 943-945Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]SpCas9-NGNGR1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337RHighly relaxed PAM[28.Hua K. et al.Genome engineering in rice using Cas9 variants that recognize NG PAM sequences.Mol. Plant. 2019; 12: 1003-1014Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,34.Zhong Z. et al.Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG.Mol. Plant. 2019; 12: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,35.Endo M. et al.Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM.Nat. Plants. 2019; 5: 14-17Crossref PubMed Scopus (72) Google Scholar,43.Qin R. et al.SpCas9-NG self-targets the sgRNA sequence in plant genome editing.Nat. Plants. 2020; 6: 197-201Crossref PubMed Scopus (15) Google Scholar]iSpymacCas9NAAR221K/N394KGood for A-rich site[36.Sretenovic S. et al.Expanding plant genome editing scope by an engineered iSpyMacCas9 system targeting the A-rich PAM sequences.Plant Commun. 2020; 2: 100101Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar]SpCas9-HF1NGGN497A/R661A/Q695A/Q926ALow off-target[145.Zhang Q. et al.Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.Plant Mol. Biol. 2018; 96: 445-456Crossref PubMed Scopus (76) Google Scholar, 146.Zhang D. et al.Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.Genome Biol. 2017; 18: 191Crossref PubMed Scopus (68) Google Scholar, 147.Xu W. et al.Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.BMC Plant Biol. 2019; 19: 511Crossref PubMed Scopus (8) Google Scholar]eSpCas9NGGK810A/K1003A/R1060ALow off-target[145.Zhang Q. et al.Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.Plant Mol. Biol. 2018; 96: 445-456Crossref PubMed Scopus (76) Google Scholar, 146.Zhang D. et al.Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.Genome Biol. 2017; 18: 191Crossref PubMed Scopus (68) Google Scholar, 147.Xu W. et al.Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.BMC Plant Biol. 2019; 19: 511Crossref PubMed Scopus (8) Google Scholar]HypaCas9NGGN692A/M694A/Q695A/H698ALow off-target[147.Xu W. et al.Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice.BMC Plant Biol. 2019; 19: 511Crossref PubMed Scopus (8) Google Scholar,148.Liang Z. et al.Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes.Plant Biotechnol. J. 2018; 16: 2053-2062Crossref PubMed Scopus (31) Google Scholar]eHF1-Cas9NGGN497A/R661A/Q695A/K848A/Q926A/K1003A/R1060ALow off-target[148.Liang Z. et al.Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes.Plant Biotechnol. J. 2018; 16: 2053-2062Crossref PubMed Scopus (31) Google Scholar]eHypa-Cas9NGGN692A/M694A/Q695A/H698A/K848A/K1003A/R1060ALow off-target[148.Liang Z. et al.Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes.Plant Biotechnol. J. 2018; 16: 2053-2062Crossref PubMed Scopus (31) Google Scholar]HiFi Cas9NGGR691ALow off-target[149.Banakar R. et al.Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS) gene.Rice (N Y). 2020; 13: 4Crossref PubMed Scopus (11) Google Scholar]xCas9NG, GAA GATA262T/R324L/S409I/E480K/E543D/M694I/E1219VLow off-target Flexible PAM[27.Ge Z. et al.Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants.Plant Biotechnol. J. 2019; 17: 1865-1867Crossref PubMed Scopus (27) Google Scholar,29.Li J. et al.Plant genome editing using xCas9 with expanded PAM compatibility.J. Genet. Genomics. 2019; 46: 277-280Crossref PubMed Scopus (12) Google Scholar,33.Wang J. et al.xCas9 expands the scope of genome editing with reduced efficiency in rice.Plant Biotechnol. J. 2019; 17: 709-711Crossref PubMed Scopus (44) Google Scholar,150.Zeng D. et al.Engineered Cas9 variant tools expand targeting scope of genome and base editing in rice.Plant Biotechnol. J. 2020; 18: 1348-1350Crossref PubMed Scopus (17) Google Scholar]SaCas9NNGRRTNatural variantLow off-target High efficiency[26.Steinert J. et al.Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus.Plant J. 2015; 84: 1295-1305Crossref PubMed Google Scholar,110.Wolter F. et al.Efficient in planta gene targeting in Arabidopsis using egg cell-specific expression of the Cas9 nuclease of Staphylococcus aureus.Plant J. 2018; 94: 735-746Crossref PubMed Scopus (56) Google Scholar]SaCas9-KKHNNNRRTE782K/N968K/R1015HFlexible PAM[151.Qin R. et al.Developing a highly efficient and wildly adaptive CRISPR-SaCas9 toolset for plant genome editing.Plant Biotechnol. J. 2019; 17: 706-708Crossref PubMed Scopus (24) Google Scholar]St1Cas9NNAGAAWNatural variantAlternate PAM[26.Steinert J. et al.Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus.Plant J. 2015; 84: 1295-1305Crossref PubMed Google Scholar]ScCas9NNGNatural variantFlexible PAM[152.Wang M. et al.Targeted base editing in rice with CRISPR/ScCas9 system.Plant Biotechnol. J. 2020; 18: 1645-1647Crossref PubMed Scopus (18) Google Scholar]XNG-Cas9R1335V/A262T/R324L/S409I/E480K/E543D/M694I/L1111R/D1135V/G1218R/E1219V/E1219F/A1322R/T1337RHighly relaxed PAM[153.Niu Q. et al.Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.J. Integr. Plant Biol. 2020; 62: 398-402Crossref PubMed Scopus (12) Google Scholar]SpRYNGD, NAND1135L/S1136W/G1218K/E1219Q/R1335Q/T1337RHighly flexible PAM[38.Ren Q. et al.PAM-less plant genome editing using a CRISPR-SpRY toolbox.Nat. Plants. 2021; 7: 25-33Crossref PubMed Scopus (0) Google Scholar,39.Xu Z. et al.SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition.Genome Biol. 2021; 22: 6Crossref PubMed Scopus (19) Google Scholar,41.Li J. et al.Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.Mol. Plant. 2021; 14: 352-360Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]SpGNGD1135L/S1136W/G1218K/E1219Q/R1335Q/T1337RHighly flexible PAMSpCas9-NRRHNRRHI322V/S409I/E427G/R654L/R753G/R1114G/D1135N/V1139A/D1180G/E1219V/Q1221H/A1320V/R1333KFlexible PAM[41.Li J. et al.Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.Mol. Plant. 2021; 14: 352-360Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]SpCas9-NRCHNRCHI322V/S409I/E427G/R654L/R753G/R1114G/D1135N/E1219V/D1332N/R1335Q/T1337N/S1338T/H1349RFlexible PAM[41.Li J. et al.Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.Mol. Plant. 2021; 14: 352-360Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]SpCas9-NRTHNRTHI322V/S409I/E427G/R654L/R753G/R1114G/D1135N/D1180G/G1218S/E1219V/Q1221H/P1249S/E1253K/P1321S/D1322G/R1335LFlexible PAM[41.Li J. et al.Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.Mol. Plant. 2021; 14: 352-360Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]AsCas12aTTTVNatural variantT-rich PAM[154.Malzahn A.A. et al.Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis.BMC Biol. 2019; 17: 9Crossref PubMed Scopus (53) Google Scholar,155.Bernabé-Orts J.M. et al.Assessment of Cas12a-mediated gene editing efficiency in plants.Plant Biotechnol. J. 2019; 17: 1971-1984Crossref PubMed Scopus (0) Google Scholar]LbCas12aTTTVNatural variantT-rich PAM[154.Malzahn A.A. et al.Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis.BMC Biol. 2019; 17: 9Crossref PubMed Scopus (53) Google Scholar,156.Schindele P. Puchta H. Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing.Plant Biotechnol. J. 2020; 18: 1118-1120Crossref PubMed Scopus (8) Google Scholar]LbCas12a-RRTYCV, CCCCG532R/K595RAlternate PAM[157.Li S. et al.Expanding the scope of CRISPR/Cpf1-mediated genome editing in rice.Mol. Plant. 2018; 11: 995-998Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,158.Zhong Z. et al.Plant genome editing using fncpf1 and lbcpf1 nucleases at redefined and altered PAM sites.Mol. Plant. 2018; 11: 999-1002Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]LbCas12a-RVRTATVG532R/K538V/Y542RAlternate PAM[157.Li S. et al.Expanding the scope of CRISPR/Cpf1-mediated genome editing in rice.Mol. Plant. 2018; 11: 995-998Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,158.Zhong Z. et al.Plant genome editing using fncpf1 and lbcpf1 nucleases at redefined and altered PAM sites.Mol. 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