Lecture 23: Mutants and Mutations 2018 - Biology

Lecture 23: Mutants and Mutations 2018

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

The CRISPR/Cas9 technique is a highly valuable tool in creating new materials for both basic and applied researches. Previously, we succeeded in effectively generating mutations in Brassica napus using an available CRISPR/Cas9 vector pKSE401, while isolation of Cas9-free mutants is laborious and inefficient. Here, we inserted a fluorescence tag (sGFP) driven by the constitutive 35S promoter into pKSE401 to facilitate a visual screen of mutants. This modified vector was named pKSE401G and tested in several dicot plant species, including Arabidopsis, B. napus, Fragaria vesca (strawberry), and Glycine max (soybean). Consequently, GFP-positive plants were readily identified through fluorescence screening in all of these species. Among these GFP-positive plants, the average mutation frequency ranged from 20.4 to 52.5% in Arabidopsis and B. napus with stable transformation, and was 90.0% in strawberry and 75.0% in soybean with transient transformation, indicating that the editing efficiency resembles that of the original vector. Moreover, transgene-free mutants were sufficiently identified in Arabidopsis in the T2 generation and B. napus in the T1 generation based on the absence of GFP fluorescence, and these mutants were stably transmissible to next generation without newly induced mutations. Collectively, pKSE401G provides us an effective tool to readily identify positive primary transformants and transgene-free mutants in later generations in a wide range of dicot plant species.

Keywords: Arabidopsis B. napus CRISPR/Cas9 genome editing soybean strawberry visual screening.


Design of the vector pKSE401G…

Design of the vector pKSE401G . (A) Schematic representation of the pKSE401G vector…

Generation of CR-rpk1 mutants in…

Generation of CR-rpk1 mutants in Arabidopsis . (A) The target sites of sgRNA1…

Visual screen and characterization of…

Visual screen and characterization of Cas9-free CR-rpk1 plants in the T2 generation. (A)…

Generation of CR-bnaarf2 mutants in…

Generation of CR-bnaarf2 mutants in B. napus . (A) Partial coding sequence alignment…

Visual screen and characterization of…

Visual screen and characterization of Cas9-free CR-bnaarf2 mutants in the T1 generation. (A)…

Phenotype and genotype of strawberry…

Phenotype and genotype of strawberry fruit transiently transformed with CR-FveMYB10. (A) The target…

Phenotype and genotype of soybean…

Phenotype and genotype of soybean roots transiently transformed with CR-GmNFR1a . (A) The…

Atypical activation of the G protein Gα q by the oncogenic mutation Q209P

The causative role of G protein-coupled receptor (GPCR) pathway mutations in uveal melanoma (UM) has been well-established. Nearly all UMs bear an activating mutation in a GPCR pathway mediated by G proteins of the Gq/11 family, driving tumor initiation and possibly metastatic progression. Thus, targeting this pathway holds therapeutic promise for managing UM. However, direct targeting of oncogenic Gαq/11 mutants, present in ∼90% of UMs, is complicated by the belief that these mutants structurally resemble active Gαq/11 WT. This notion is solidly founded on previous studies characterizing Gα mutants in which a conserved catalytic glutamine (Gln-209 in Gαq) is replaced by leucine, which leads to GTPase function deficiency and constitutive activation. Whereas Q209L accounts for approximately half of GNAQ mutations in UM, Q209P is as frequent as Q209L and also promotes oncogenesis, but has not been characterized at the molecular level. Here, we characterized the biochemical and signaling properties of Gαq Q209P and found that it is also GTPase-deficient and activates downstream signaling as efficiently as Gαq Q209L. However, Gαq Q209P had distinct molecular and functional features, including in the switch II region of Gαq Q209P, which adopted a conformation different from that of Gαq Q209L or active WT Gαq, resulting in altered binding to effectors, Gβγ, and regulators of G-protein signaling (RGS) proteins. Our findings reveal that the molecular properties of Gαq Q209P are fundamentally different from those in other active Gαq proteins and could be leveraged as a specific vulnerability for the ∼20% of UMs bearing this mutation.

Keywords: G protein-coupled receptor (GPCR) GNAQ GTPase cancer biology cell signaling heterotrimeric G protein melanoma oncogenesis signal transduction uveal melanoma.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Human leukemia mutations corrupt but do not abrogate GATA-2 function

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 -77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin - Kit + myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2-linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2-dependent pathologies.

Keywords: AML GATA-2 MDS hematopoiesis leukemia.

Conflict of interest statement

The authors declare no conflict of interest.


GATA-2 N-finger leukemia mutations attenuate…

GATA-2 N-finger leukemia mutations attenuate chromatin occupancy and target gene activation. ( A…

Structural determinants of GATA-2 function.…

Structural determinants of GATA-2 function. ( A , Upper ) Schematic representation of…

GATA-2 leukemia mutants increase myeloid…

GATA-2 leukemia mutants increase myeloid progenitor cell activity in a primary cell genetic…

GATA-2 leukemia mutants stimulate myelopoiesis…

GATA-2 leukemia mutants stimulate myelopoiesis ex vivo. ( A ) Schematic representation of…

GATA-2 leukemia mutant increases cell…

GATA-2 leukemia mutant increases cell cycle progression in a genetic complementation assay. (…

GATA-2 leukemia mutations: gain-of-function and…

GATA-2 leukemia mutations: gain-of-function and loss-of-function consequences. ( A ) Molecular activities of…


The growth-repressing function of ABA has been thought to be a trade-off for improving stress adaptation (3, 33, 34). Since the plant growth and stress adaptation processes are contrary to each other in many ways, it is important to establish a balance between them that is appropriate for the environment. Therefore, ABA levels and signaling should have significant effects on both growth and adaptation. Genetic adjustment of the balance by manipulating ABA levels and/or signaling may generate useful crop varieties to improve productivity in specific environments.

The PYLs are currently the largest plant hormone receptor family known (35). In Arabidopsis, 14 PYL members have been identified, and redundant as well as differential functions of the genes have been documented (11, 26 ⇓ –28, 35, 36). In rice, 13 PYL members were predicted (29, 30). Our results indicate differential and redundant functions for different members as well. The functional differentiation and redundancy provide the possibility to adjust the balance between growth and stress resistance to improve crop productivity through editing certain PYLs. In rice, we found that among the pyl mutants, pyl1/4/6 showed the best growth, while maintaining nearly normal seed dormancy. During the heat wave of the 2016 summer in the paddy field in Shanghai, although the growth of quintuple to septuple pyl mutants was increasingly retarded, the pyl1/4/6 mutants still grew better than the wild type. This suggests that the pyl1/4/6 lines may be more tolerant to the hot weather than the quintuple, sextuple, and septuple group I mutants. These results indicate that the balance between growth and stress adaptation is altered in high-order group I pyl mutants, and that the new balance in pyl1/4/6 plants favors more growth, while stress adaptation is less compromised than in higher order group I pyl mutants.

Previous studies showed that Arabidopsis high-order pyl mutants exhibited retarded growth, and that this growth defect can be ameliorated by increasing the humidity of the growth environment (25, 37). Still, improved growth (compared with the wild type) was not observed in Arabidopsis pyl mutants (25). Although, overall, rice PYLs have similar functions to Arabidopsis PYLs in promoting seed dormancy and stomatal closure, they seem to differ in their impacts on plant growth. It is likely that under the paddy field growth conditions, some of the rice PYLs have been selected to have a particularly important role in restraining plant growth. In the future, it will be of interest to determine how PYL1, PYL4, and PYL6 (particularly PYL6) are linked to growth regulation in rice. In the paddy field, where water is not limiting, the higher transpiration of pyl1/4/6 may contribute to faster growth and higher yield by facilitating rapid CO2 absorption, while avoiding the negative effects of excessive water loss. Plant breeders have long suggested that the improvement of crop performance in certain climates may be achieved by reducing responses to ABA or endogenous ABA level (34). The pyl1/4/6 rice mutants may represent an effective approach to achieve this historically coveted result.

Most mutations that cause spinocerebellar ataxia autosomal recessive type 16 (SCAR16) destabilize the protein quality-control E3 ligase CHIP

The accumulation of misfolded proteins promotes protein aggregation and neuronal death in many neurodegenerative diseases. To counteract misfolded protein accumulation, neurons have pathways that recognize and refold or degrade aggregation-prone proteins. One U-box-containing E3 ligase, C terminus of Hsc70-interacting protein (CHIP), plays a key role in this process, targeting misfolded proteins for proteasomal degradation. CHIP plays a protective role in mouse models of neurodegenerative disease, and in humans, mutations in CHIP cause spinocerebellar ataxia autosomal recessive type 16 (SCAR16), a fatal neurodegenerative disease characterized by truncal and limb ataxia that results in gait instability. Here, we systematically analyzed CHIP mutations that cause SCAR16 and found that most SCAR16 mutations destabilize CHIP. This destabilization caused mutation-specific defects in CHIP activity, including increased formation of soluble oligomers, decreased interactions with chaperones, diminished substrate ubiquitination, and reduced steady-state levels in cells. Consistent with decreased CHIP stability promoting its dysfunction in SCAR16, most mutant proteins recovered activity when the assays were performed below the mutants' melting temperature. Together, our results have uncovered the molecular basis of genetic defects in CHIP function that cause SCAR16. Our insights suggest that compounds that improve the thermostability of genetic CHIP variants may be beneficial for treating patients with SCAR16.

Keywords: molecular chaperone neurodegenerative disease protein misfolding proteostasis ubiquitin ligase.

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article


Hodgkinson A, Eyre-Walker A. Variation in the mutation rate across mammalian genomes. Nat Rev Genet. 201112:756–66.

Schuster-Bockler B, Lehner B. Chromatin organization is a major influence on regional mutation rates in human cancer cells. Nature. 2012488:504–7.

Frigola J, Sabarinathan R, Mularoni L, Muinos F, Gonzalez-Perez A, Lopez-Bigas N. Reduced mutation rate in exons due to differential mismatch repair. Nat Genet. 201749:1684–92.

Prendergast JG, Campbell H, Gilbert N, Dunlop MG, Bickmore WA, Semple CA. Chromatin structure and evolution in the human genome. BMC Evol Biol. 20077:72.

Lang GI, Murray AW. Mutation rates across budding yeast chromosome VI are correlated with replication timing. Genome Biol Evol. 20113:799–811.

Stamatoyannopoulos JA, Adzhubei I, Thurman RE, Kryukov GV, Mirkin SM, Sunyaev SR. Human mutation rate associated with DNA replication timing. Nat Genet. 200941:393–5.

Chen CL, Rappailles A, Duquenne L, Huvet M, Guilbaud G, Farinelli L, Audit B, d'Aubenton-Carafa Y, Arneodo A, Hyrien O, Thermes C. Impact of replication timing on non-CpG and CpG substitution rates in mammalian genomes. Genome Res. 201020:447–57.

Weber CC, Pink CJ, Hurst LD. Late-replicating domains have higher divergence and diversity in Drosophila melanogaster. Mol Biol Evol. 201229:873–82.

Supek F, Lehner B. Differential DNA mismatch repair underlies mutation rate variation across the human genome. Nature. 2015521:81–4.

Herman RK, Dworkin NB. Effect of gene induction on the rate of mutagenesis by ICR-191 in Escherichia coli. J Bacteriol. 1971106:543–50.

Park C, Qian W, Zhang J. Genomic evidence for elevated mutation rates in highly expressed genes. EMBO Rep. 201213:1123–9.

Savic DJ, Kanazir DT. The effect of a histidine operator-constitutive mutation on UV-induced mutability within the histidine operon of Salmonella typhimurium. Mol Gen Genet. 1972118:45–50.

Chen X, Yang JR, Zhang J. Nascent RNA folding mitigates transcription-associated mutagenesis. Genome Res. 201626:50–9.

Hanawalt PC, Spivak G. Transcription-coupled DNA repair: two decades of progress and surprises. Nat Rev Mol Cell Biol. 20089:958–70.

Coulondre C, Miller JH, Farabaugh PJ, Gilbert W. Molecular basis of base substitution hotspots in Escherichia coli. Nature. 1978274:775–80.

Aggarwala V, Voight BF. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome. Nat Genet. 201648:349–55.

Zhu YO, Siegal ML, Hall DW, Petrov DA. Precise estimates of mutation rate and spectrum in yeast. Proc Natl Acad Sci U S A. 2014111:E2310–8.

Blake RD, Hess ST, Nicholson-Tuell J. The influence of nearest neighbors on the rate and pattern of spontaneous point mutations. J Mol Evol. 199234:189–200.

Olson WK, Gorin AA, Lu XJ, Hock LM, Zhurkin VB. DNA sequence-dependent deformability deduced from protein-DNA crystal complexes. Proc Natl Acad Sci U S A. 199895:11163–8.

Harteis S, Schneider S. Making the bend: DNA tertiary structure and protein-DNA interactions. Int J Mol Sci. 201415:12335–63.

Rohs R, West SM, Sosinsky A, Liu P, Mann RS, Honig B. The role of DNA shape in protein-DNA recognition. Nature. 2009461:1248–53.

Wang X, Zhou T, Wunderlich Z, Maurano MT, DePace AH, Nuzhdin SV, Rohs R. Analysis of genetic variation indicates DNA shape involvement in purifying selection. Mol Biol Evol. 201835:1958–67.

Lang GI, Murray AW. Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae. Genetics. 2008178:67–82

Lee W, Tillo D, Bray N, Morse RH, Davis RW, Hughes TR, Nislow C. A high-resolution atlas of nucleosome occupancy in yeast. Nat Genet. 200739:1235–44.

Bolshoy A, McNamara P, Harrington RE, Trifonov EN. Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles. Proc Natl Acad Sci U S A. 199188:2312–6.

Wolfe KH, Sharp PM, Li WH. Mutation rates differ among regions of the mammalian genome. Nature. 1989337:283–5.

Chen X, Chen Z, Chen H, Su Z, Yang J, Lin F, Shi S, He X. Nucleosomes suppress spontaneous mutations base-specifically in eukaryotes. Science. 2012335:1235–8.

Smith T, Ho G, Christodoulou J, Price EA, Onadim Z, Gauthier-Villars M, Dehainault C, Houdayer C, Parfait B, van Minkelen R, et al. Extensive variation in the mutation rate between and within human genes associated with Mendelian disease. Hum Mutat. 201637:488–94.

Bouaoun L, Sonkin D, Ardin M, Hollstein M, Byrnes G, Zavadil J, Olivier M. TP53 variations in human cancers: new lessons from the IARC TP53 database and genomics data. Hum Mutat. 201637:865–76.

Fares MA, Keane OM, Toft C, Carretero-Paulet L, Jones GW. The roles of whole-genome and small-scale duplications in the functional specialization of Saccharomyces cerevisiae genes. PLoS Genet. 20139:e1003176

Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD, Varela I, Lin ML, Ordonez GR, Bignell GR, et al. A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 2010463:191–6.

Cancer Genome Atlas Research Network, Weinstein JN, Collisson EA, Mills GB, Shaw KR, Ozenberger BA, Ellrott K, Shmulevich I, Sander C, Stuart JM. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. 201345:1113–20.

Capuano F, Mulleder M, Kok R, Blom HJ, Ralser M. Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species. Anal Chem. 201486:3697–702.

Jonsson H, Sulem P, Kehr B, Kristmundsdottir S, Zink F, Hjartarson E, Hardarson MT, Hjorleifsson KE, Eggertsson HP, Gudjonsson SA, et al. Parental influence on human germline de novo mutations in 1,548 trios from Iceland. Nature. 2017549:519–22

Hagerman PJ. Sequence-directed curvature of DNA. Nature. 1986321:449–50.

Koo HS, Wu HM, Crothers DM. DNA bending at adenine. Thymine tracts. Nature. 1986320:501–6.

Ulanovsky LE, Trifonov EN. Estimation of wedge components in curved DNA. Nature. 1987326:720–2.

Luria SE, Delbruck M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics. 194328:491–511.

Maki H. Origins of spontaneous mutations: specificity and directionality of base-substitution, frameshift, and sequence-substitution mutagenesis. Annu Rev Genet. 200236:279–303.

Alexandrov LB, Nik-Zainal S, Wedge DC, Campbell PJ, Stratton MR. Deciphering signatures of mutational processes operative in human cancer. Cell Rep. 20133:246–59.

Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, Carter SL, Stewart C, Mermel CH, Roberts SA, et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature. 2013499:214–8.

Polak P, Karlic R, Koren A, Thurman R, Sandstrom R, Lawrence M, Reynolds A, Rynes E, Vlahovicek K, Stamatoyannopoulos JA, Sunyaev SR. Cell-of-origin chromatin organization shapes the mutational landscape of cancer. Nature. 2015518:360–4.

Hurst LD, Smith NG. Do essential genes evolve slowly? Curr Biol. 19999:747–50.

Hirsh AE, Fraser HB. Protein dispensability and rate of evolution. Nature. 2001411:1046–9.

Wang Z, Zhang J. Why is the correlation between gene importance and gene evolutionary rate so weak? PLoS Genet. 20095:e1000329.

Liao BY, Scott NM, Zhang J. Impacts of gene essentiality, expression pattern, and gene compactness on the evolutionary rate of mammalian proteins. Mol Biol Evol. 200623:2072–80.

Zhang J, Yang JR. Determinants of the rate of protein sequence evolution. Nat Rev Genet. 201516:409–20.

Li C, Qian W, Maclean CJ, Zhang J. The fitness landscape of a tRNA gene. Science. 2016352:837–40.

Puchta O, Cseke B, Czaja H, Tollervey D, Sanguinetti G, Kudla G. Network of epistatic interactions within a yeast snoRNA. Science. 2016352:840–4.

He X, Liu L. EVOLUTION. Toward a prospective molecular evolution. Science. 2016352:769–70.

Wal M, Pugh BF. Genome-wide mapping of nucleosome positions in yeast using high-resolution MNase ChIP-Seq. Methods Enzymol. 2012513:233–50.

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 20129:357–9.

Wang Y, Song F, Zhu J, Zhang S, Yang Y, Chen T, Tang B, Dong L, Ding N, Zhang Q, et al. GSA: genome sequence archive. Genomics Proteomics Bioinformatics. 201715:14–8.

Lorenz R, Bernhart SH, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, Hofacker IL. ViennaRNA Package 2.0. Algorithms Mol Biol. 20116:26.

Chen S, Li K, Cao W, Wang J, Zhao T, Huan Q, Yang YF, Wu S, Qian W. Codon-resolution analysis reveals a direct and context-dependent impact of individual synonymous mutations on mRNA level. Mol Biol Evol. 201734:2944–58.

Christie KR, Weng S, Balakrishnan R, Costanzo MC, Dolinski K, Dwight SS, Engel SR, Feierbach B, Fisk DG, Hirschman JE, et al. Saccharomyces genome database (SGD) provides tools to identify and analyze sequences from Saccharomyces cerevisiae and related sequences from other organisms. Nucleic Acids Res. 200432:D311–4.

Deutschbauer AM, Jaramillo DF, Proctor M, Kumm J, Hillenmeyer ME, Davis RW, Nislow C, Giaever G. Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast. Genetics. 2005169:1915–25

Qian W, Ma D, Xiao C, Wang Z, Zhang J. The genomic landscape and evolutionary resolution of antagonistic pleiotropy in yeast. Cell Rep. 20122:1399–410

Qian W, Zhang J. Genomic evidence for adaptation by gene duplication. Genome Res. 201424:1356–62

Chatr-Aryamontri A, Oughtred R, Boucher L, Rust J, Chang C, Kolas NK, O'Donnell L, Oster S, Theesfeld C, Sellam A, et al. The BioGRID interaction database: 2017 update. Nucleic Acids Res. 201745:D369–79.

Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, Mis M, Zimmermann M, Fradet-Turcotte A, Sun S, et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell. 2015163:1515–26

Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, Lander ES, Sabatini DM. Identification and characterization of essential genes in the human genome. Science. 2015350:1096–101

Huang N, Lee I, Marcotte EM, Hurles ME. Characterising and predicting haploinsufficiency in the human genome. PLoS Genet. 20106:e1001154

Friedel M, Nikolajewa S, Suhnel J, Wilhelm T. DiProDB: a database for dinucleotide properties. Nucleic Acids Res. 200937:D37–40.

Rehm HL, Berg JS, Brooks LD, Bustamante CD, Evans JP, Landrum MJ, Ledbetter DH, Maglott DR, Martin CL, Nussbaum RL, et al. ClinGen--the clinical genome resource. N Engl J Med. 2015372:2235–42.

Duan C, Huan Q, Chen X, Wu S, Carey LB, He X, Qian W. Reduced intrinsic DNA curvature leads to increased mutation rate. Genome Sequence Archive. 2018. The release date: 19 Apr 2018.

The protein LZTR1 is mutated in human cancers and developmental diseases. Work from two groups now converges to implicate the protein in regulating signaling by the small guanosine triphosphatase RAS. Steklov et al. showed that mice haploinsufficient for LZTR1 recapitulated aspects of the human disease Noonan syndrome. Their biochemical studies showed that LZTR1 associated with RAS. LZTR1 appears to function as an adaptor that promotes ubiquitination of RAS, thus inhibiting its signaling functions. Bigenzahn et al. found LZTR1 in a screen for proteins whose absence led to resistance to the tyrosine kinase inhibitors used to treat cancers caused by the BCR-ABL oncogene product. Their biochemical studies and genetic studies in fruitflies also showed that loss of LZTR1 led to increased activity of RAS and signaling through the mitogen-activated protein kinase pathway.

The leucine zipper–like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

Mutations concurrent with loss of heterozygosity at leucine zipper–like transcriptional regulator 1 (LZTR1) are associated with glioblastoma and schwannomatosis (13). LZTR1 mutations predispose for pediatric neoplasms and are increased over background in liver and testicular cancers (4, 5). The most recurrent LZTR1 mutation in cancer is an inactivating splice-site mutation at codon 217 (fig. S1) (4, 6). LZTR1 constitutes to Noonan syndrome caused by dysregulation of the guanosine triphosphatase RAS (79). However, how LZTR1 contributes to human disease is not known.

To uncover Lztr1 disease mechanisms, we used an Lztr1 deletion mouse model. We found that loss of Lztr1 is lethal between embryonic day 17.5 (E17.5) and birth (fig. S2A). Lztr1 +/− male mice exhibited decreased weight (fig. S2, B to D) and facial dysmorphia (Fig. 1A). Lztr1 +/− mice, both male and female, displayed heart malformations, including decreased left ventricular systolic function, increased diastolic dimensions, eccentric hypertrophy, increased cardiomyocyte area, and reduced longevity (Fig. 1, B and C, and fig. S2, E and F). Collectively, our results show that Lztr1 +/− mice recapitulate some phenotypes of human Noonan syndrome patients, indicating that LZTR1 function is evolutionary conserved.

(A) Morphometric characteristics of the skulls of 12-month-old Lztr1 +/+ and Lztr1 +/− male mice. (B) Haematoxylin and eosin–stained heart ventricular sections. Scale bar, 0.5 mm. The total cardiac area was quantified by Fiji. In the graph, horizontal lines represent means ± SD. (C) A mean area of 200 cardiomyocytes measured in laminin-stained heart sections of Lztr1 +/+ and Lztr1 +/− mice. Horizontal lines represent means ± SD. (D) Growth rate of early-passage MEFs isolated from three Lztr1 +/+ and three Lztr1 −/− embryos. (E) Growth rate of Lztr1 −/− MEFs expressing an empty vector (EV), wt-LZTR1, or LZTR1 mutants. n = 3. (F) AI growth of Schwann cells expressing Cas9 or Cas9/gLZTR1 (gLZTR1, guide RNA targeting LZTR1). n = 3. (G) AI growth of LZTR1-indel Schwann cells expressing the indicated constructs. n = 3. M202R, Met 202 →Arg. (H) Quantitative real time polymerase chain reaction (qRT-PCR) analysis of mRNA expression in primary human Schwann cells expressing shGFP or pooled shLZTR1. n = 3. For (D) to (H), values are means ± SEM. For (A) to (C) and (F) to (H), P values are from a two-sided Student’s t test. For (D) and (E), P values were detected by two-way analysis of variance (ANOVA).

We engineered several cellular models of LZTR1 loss: mouse embryo fibroblasts (MEFs) derived from Lztr1 +/+ and Lztr1 −/− mouse embryos, primary human Schwann cells expressing short hairpin green fluorescent protein (shGFP) or shLZTR1, and immortalized human Schwann cells and HeLa cells with CRISPR-Cas9–mediated LZTR1-indels (fig. S3). In all tested models, LZTR1 loss increased growth rate (Fig. 1D and fig. S4, A to C). Overexpression of wild-type LZTR1 (wt-LZTR1), but not of LZTR1 mutants, reduced the enhanced growth rate (Fig. 1E and fig. S4, D and E). Loss of LZTR1 in Schwann cells enhanced two-dimensional colony and anchorage-independent (AI) growth (Fig. 1F and fig. S4, F to H), and overexpression of wt-LZTR1, but not of disease-associated LZTR1 mutants, suppressed AI growth in LZTR1-indel cells (Fig. 1G). Furthermore, depletion of LZTR1 in Schwann cells showed a gene expression signature (Fig. 1H) resembling that of proliferating Schwann cells during nerve regeneration (10). These data suggest that LZTR1 loss drives Schwann cells from quiescent, myelinating cells into proliferating cells.

LZTR1 acts a substrate adaptor for cullin 3 (CUL3) ubiquitin ligase complexes (11). To identify candidate LZTR1 substrates, we used a mass spectrometry (MS) Virotrap method, which allowed the trapping of protein complexes from intact mammalian cells (fig. S5A) (12). The screen with LZTR1 as bait detected CUL3, Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma RAS viral oncogene homolog (NRAS) among the top hits (Fig. 2A). The reciprocal Virotrap screen with the HRAS-deltaCAAX mutant, which lacks the last four amino acids, confirmed the complex formation with LZTR1 and identified CUL3 (Fig. 2B). Furthermore, a panRAS antibody that recognizes all RAS isoforms coimmunoprecipitated with hemagglutinin (HA)–tagged LZTR1. Similarly, Flag-tagged LZTR1 coimmunoprecipitated with endogenous RAS proteins, but not RAC1 (fig. S5B). Moreover, we introduced a Halo-tag HiBiT (13) to the LZTR1 locus in HeLa cells and MEFs (Fig. 2C and fig. S5, C and D). panRAS antibody coimmunoprecipitated with endogenous RAS and endogenous HiBiT-LZTR1 (Fig. 2C and fig. S5E). Reciprocal coimmunoprecipitations (co-IPs) demonstrated that LZTR1 interacted with each of the three Flag-RAS isoforms (fig. S5F). Together, these results indicate that LZTR1, CUL3, and RAS form a complex.

(A and B) Virotrap screens performed in HEK293T cells using group-specific antigen (GAG)–LZTR1 (A) or GAG-HRAS–deltaCAAX (deltaC) (B) as baits. Escherichia coli dihydrofolate reductase (eDHFR) fused to GAG was used as a negative control. (C) A scheme for the generation of cells expressing in-frame HiBiT-LZTR1 protein. RAS was immunoprecipitated from the HiBiT-LZTR1 edited HeLa cell lysates with panRAS antibody. Luminescent signal was generated by HiBiT incubated with LgBiT. nt, nucleotide. (D) Ubiquitinated RAS was purified from HEK293T cells expressing the indicated constructs by Co 2+ metal affinity chromatography and detected by immunoblotting. Numbers on the left are molecular masses in kDa. 2xUb, two Ub 1xUb, one Ub EV, empty vector, WCL, whole-cell lysate ns, nonspecific. (E) A workflow for the ubiquitome analysis. LC-MS/MS, liquid chromatography–tandem mass spectrometry. (F) A heatmap showing differentially ubiquitinated peptides in Lztr1 +/+ and Lztr1 −/− MEFs. The scale shows Z-scored site intensity values. (G) The quantification of the PLA analysis of Schwann cells expressing Cas9 or Cas9/gLZTR1 using antibodies against panRAS and Ub. Values are means ± SEM n = 3. P values are from a two-sided Student’s t test. Single-letter abbreviations for the amino acid residues are as follows: A, Ala C, Cys D, Asp E, Glu F, Phe G, Gly H, His I, Ile K, Lys L, Leu M, Met N, Asn P, Pro Q, Gln R, Arg S, Ser T, Thr V, Val W, Trp and Y, Tyr.

To test whether the LZTR1-CUL3 complex might control RAS ubiquitination, we performed an in vitro ubiquitination reaction. We observed ubiquitination of wt-HRAS specifically in the presence of the LZTR1-CUL3 complex (fig. S6A). Coexpression of LZTR1 and CUL3 in human embryonic kidney (HEK) 293T cells increased amounts of ubiquitinated RAS (Fig. 2D and fig. S6B). By contrast, treatment with the cullin neddylation inhibitor MLN4924 or loss of LZTR1 led to decreased ubiquitination of all Flag-tagged RAS protein isoforms (fig. S6, C to F). Thus, the LZTR1-CUL3 complex can promote ubiquitination of RAS.

To investigate the role of LZTR1 in ubiquitination of endogenous RAS, we characterized ubiquitination profiles of Lztr1 +/+ and Lztr1 −/− MEFs by MS (Fig. 2E). Ubiquitome analysis revealed that ubiquitination of Hras at Lys 170 (K170) was abrogated in MEFs lacking Lztr1 (Fig. 2F and fig. S6G), indicating that endogenous Ras may serve as a substrate for the LZTR1-CUL3 complex. We also optimized a proximity ligation assay (PLA) with paired antibodies to ubiquitin (Ub) and panRAS. Consistent with the MS results, Hras-K170R (Lys 170 →Arg) knock-in or depletion of LZTR1 led to a decrease in panRAS-Ub proximity signals (Fig. 2G and fig. S7). MS analyses failed to detect any C-terminal peptides of Nras or Kras, perhaps because these isoforms are not highly expressed in MEFs and their C termini are lysine-rich (fig. S8A). However, LZTR1 interacted with (fig. S5F) and ubiquitinated all three RAS isoforms (fig. S6F), consistent with evolutionary conservation of K170 (fig. S8D). Thus, the LZTR1-CUL3 complex appears to mediate ubiquitination of all RAS isoforms.

Although multiple truncating and missense mutations of LZTR1 have been reported in Noonan syndrome and schwannomatosis (13, 14), no recurrent germline LZTR1 mutations have been identified to date. Additional sequencing analysis of blood samples from schwannomatosis patients revealed several recurrent germline mutations of LZTR1 within the BTB (broad-complex, tramtrack, and bric-a-brac)–BACK domains predicted to mediate dimerization and CUL3 binding (11, 15) (Fig. 3A). Concordantly, the BTB-BACK LZTR1 mutants, except L812P (Leu 812 →Pro), exhibited reduced binding to CUL3 (Fig. 3B and fig. S9A). Although LZTR1-L812P retained interaction with CUL3, it failed to form dimers (Fig. 3C). Oligomerization of BTB domains determines the subcellular distribution of CUL3 adaptors (15, 16). Indeed, both endogenous and ectopically expressed HA-tagged LZTR1 showed punctate endomembrane immunostaining (fig. S9B), whereas the BTB-BACK domain LZTR1 mutants, including LTZR1-L812P, showed diffuse cytoplasmic staining (Fig. 3D and fig. S9C).

(A) LZTR1 mutations in schwannomatosis and Noonan syndrome individuals. Missense LZTR1 mutations identified in our cohort of schwannomatosis patients are shown in blue. In the schematic, K indicates Kelch domain. See Fig. 2 legend for amino acid abbreviations. (B) Flag-tagged CUL3 purified from HEK293T cells was incubated with HA-tagged LZTR1-overexpressing cell lysates and then immunoprecipitated using anti-Flag resin. LZTR1 was detected by immunoblotting with anti-HA antibody. (C) Cross-linking reactions were performed using HA-tagged LZTR1 purified from HEK293T cells. LZTR1 was detected by immunoblotting using anti-HA antibody. (D) Immunostaining of HeLa cells expressing HA-tagged wt-LZTR1 or LZTR1 mutants with anti-HA antibody. Scale bar, 10 μm. (E) RAS proteins were immunoprecipitated with antibody against panRAS. LZTR1 was detected by immunoblotting with anti-HA antibody. (F) Colocalization of mCherry-NRAS and HA-tagged LZTR1 expressed in HeLa-Cas9/gLZTR1 cells. Values are means ± SEM. P values were detected by two-sided Student’s t test. (G) Ubiquitinated NRAS was purified from HEK293T cells expressing the indicated constructs by Co 2+ metal affinity chromatography and detected by anti-Flag antibody.

Missense mutations within the LZTR1 Kelch domain predicted to mediate substrate binding are also found in human disease. In co-IP assays, Kelch domain LZTR1 mutants showed decreased binding to RAS (Fig. 3E). The Kelch domain mutants, like wt-LZTR1, displayed punctate immunostaining, but only wt-LZTR1 led to relocalization of RAS to the LZTR1-CUL3–containing puncta, which represent loci of LZTR1-CUL3–mediated ubiquitination (Fig. 3F and fig. S9, D and E). Consistently, the LZTR1-L812P mutant, which does not form puncta, only weakly ubiquitinated RAS, as did the LZTR1-Y726* (Tyr 726 →Stop) mutant (Fig. 3G). Thus, disease-associated LZTR1 mutations appear to abrogate RAS ubiquitination by disrupting the formation of the RAS-LZTR1-CUL3 complex.

RAS ubiquitination affects RAS–mitogen-activated protein kinase (MAPK) signaling (17). Loss of LZTR1 led to increased RAS activity and phosphorylation of MEK1/MEK2 and ERK1/ERK2 in all tested model systems, whereas enhanced phosphorylation of V-Akt murine thymoma viral oncogene homolog (AKT) was cell dependent (Fig. 4A and fig. S10). After serum stimulation, Lztr1 −/− MEFs showed higher MEK1/MEK2 activity at all time points, whereas Lztr1 +/− MEFs had higher MEK1/MEK2 phosphorylation only at later time points (fig. S11A). Thus, Lztr1 abundance may fine-tune the activation of Ras signaling. Restoration of wt-LZTR1 expression in LZTR1-indel cells decreased MEK1/MEK2 activity (fig. S11B). Finally, LZTR1-mutated schwannomas showed strong staining of phosphorylated ERK1/ERK2 compared to wt-LZTR1 nerve trunk (fig. S11, C and D). The MEK1 inhibitor pimasertib abolished the colony growth difference between wt-LZTR1 and LZTR1-mutant cells (fig. S11E). Pimasertib treatment also rescued the embryonic lethality of Lztr1 −/− mice (Fig. 4B). Thus, LZTR1-mediated phenotypes arise, at least in part, from increased RAS signaling.

(A) MEFs isolated from three Lztr1 +/+ and three Lztr1 −/− embryos were serum-starved, stimulated with 10% serum, and analyzed by immunoblotting. Values are means of phosphorylated (p) relative to nonphosphorylated protein levels ± SEM. P values are from a two-way ANOVA. (B) Progeny from the indicated Lztr1 +/− matings. Pregnant mice were treated with pimasertib starting from E7.5. (C) Quantitative ubiquitome analysis of Lztr1 +/+ and Lztr1 −/− MEFs. FDR, false discovery rate. (D) Ubiquitinated RAS was purified from HEK293T cells expressing the indicated constructs by Co 2+ metal affinity chromatography and detected by immunoblotting. (E) The PLA analysis of wt-Hras and Hras-K170R MEFs expressing shGFP or shLztr1 using antibodies against panRAS and Ub. Red, PLA signal blue, 4′,6-diamidino-2-phenylindole scale bar, 10 μm. (F) HEK293T cells expressing the indicated constructs were serum-starved overnight, stimulated with 10% serum, and analyzed by immunoblotting. Values are means of phosphorylated relative to nonphosphorylated protein levels ± SEM n = 3. P values are from a two-way ANOVA. (G) Snapshots of Ub-conjugated RAS at the lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids (3:1 molar ratio). (H and I) Immunoblotting of the membrane and cytoplasmic fractions isolated from HeLa cells expressing shGFP and shLZTR1 (H) or wt-Hras and Hras-K170R MEFs (I). HSP70, heat shock protein 70.

Although our MS analysis detected Ras ubiquitination at several lysines, loss of Lztr1 abrogated ubiquitination of Ras only at K170 (Fig. 4C). Thus, ubiquitination of Hras at K170 may specifically require Lzrt1. Indeed, though LZTR1 depletion hindered ubiquitination of wt-HRAS, it did not affect ubiquitination of the HRAS-K170R mutant (Fig. 4D). Loss of LZTR1 also abolished the difference in Ras ubiquitination between wt-Hras and Hras-K170R MEFs (Fig. 4E and fig. S12A). Nonetheless, the LZTR1-CUL3 complex did ubiquitinate mutant HRAS-K170R in vitro (fig. S6A). The site specificity in vivo could be directed by anchoring of RAS to the membrane. Moreover, overexpression of the HRAS-K170R mutant led to higher activation of ERK1/ERK2 than did overexpression of wt-HRAS, and LZTR1 depletion did not affect ERK1/ERK2 activity in cells overexpressing HRAS-K170R (Fig. 4F and fig. S12B). K170R knock-in MEFs also showed increased MAPK signaling and growth rates (fig. S12, C and D). Collectively, these data indicate that LZTR1-mediated ubiquitination of RAS at K170 suppresses RAS-MAPK signaling.

Ubiquitination of RAS can inhibit its activity by triggering its degradation (18). However, quantitative MS analysis did not reveal an increase in RAS protein abundance in Lztr1 −/− MEFs (fig. S6G). wt-LZTR1 and LZTR1-indel Schwann cells treated with the protein synthesis inhibitor cycloheximide also showed similar RAS stability (fig. S12E), Thus, LZTR1 regulates RAS by a nondegradative mechanism. Ubiquitination of RAS also induces its relocalization to endomembranes (19, 20). However, LZTR1 overexpression increased the endomembrane fraction of both wt-RAS and the HRAS-K170R mutant (fig. S12F). LZTR1 alone also only slightly increased RAS ubiquitination (Fig. 2D) and did not affect the MAPK pathway (fig. S12G), suggesting that LZTR1 overexpression promotes endomembrane localization of RAS independently of its ability to mediate ubiquitination at K170.

To assess how ubiquitination of RAS at K170 controls its activity, we elucidated modes of the interaction between RAS and conjugated Ub. LZTR1 colocalized with NRAS at RAB11–Transferrin receptor–positive recycling endosomes (fig. S13), suggesting that LZTR1 regulates ubiquitination of farnesylated and palmitoylated RAS. Therefore, we performed molecular simulations on lipidated RAS. In the initial structures of Ub conjugated to K170 of RAS, the hypervariable regions (HVRs) of RAS exposed their anchor portions to the solution. The long-lasting simulations showed that Ub secured the anchor portion of the HVR by sequestering the farnesyl and palmitoyl groups (fig. S14). Concordantly to a rapid kinetics of spontaneous insertion of lipidated RAS into the membrane (2124), the HVRs of nonubiquitinated RAS straightforwardly associated with membranes. However, Ub conjugation to K170 of RAS prevented the HVRs from binding to and inserting into membranes (Fig. 4G). Thus, ubiquitination at K170 may disrupt the association of RAS to the membrane. Indeed, loss of LZTR1 or Hras-K170R knock-in increased the fraction of membrane-bound RAS (Fig. 4, H and I, and fig. S15). These results are all consistent with RAS ubiquitination at K170 inhibiting RAS activity by impairing its association with the membrane.

Our results indicate that LZTR1-mediated ubiquitination of RAS on K170 modulates RAS activity, dysregulation of which leads to human disease. An accompanying study shows that LZTR1 dysregulation also confers drug resistance (25). Understanding this unconventional mechanism of RAS activation may help to identify patients who might benefit from RAS pathway inhibitors and inform new therapeutic approaches for these patients.

Genetic Insights into Mammalian Cytoplasmic Dynein Function Provided by Novel Mutations in the Mouse

Anna Kuta , . Elizabeth M.C. Fisher , in Dyneins , 2012

18.10 Using Mouse Genetics to Further Unravel the Role of the Cytoplasmic Dynein Heavy Chain

Mouse mutants have given and will continue to give novel insight into the role of cytoplasmic dynein, insights that would have been impossible to gain from in vitro studies. In addition, one aspect of working with genetic mouse models that can give information about the interactions of proteins of interest is to cross different mouse strains and analyze the phenotype of the resulting progeny. In 2005, heterozygous Dync1h1 Loa/+ mice were crossed with the SOD1 G93A transgenic mice [16] . The latter strain (TgSOD1 G93A ) carries a human transgene array of a mutant superoxide dismutase 1 gene, which is causative for ALS in both humans and mice. TgSOD1 G93A mice typically die at about 125 days of age (depending on genetic background) from motor neuron loss and with pathophysiological symptoms resembling ALS in humans.

Somewhat surprisingly, the double mutant progeny that carried the human transgene array and the Loa mutation in Dync1h1 lived for 28% longer than their TgSOD1 G93A littermates or parents. The double mutants had a delay in disease onset and death, although a similar time course of disease. Axonal transport was analysed in embryonic motor neurons derived from progeny of the cross, using a fluorescently labeled fragment of the tetanus toxin. This showed two surprising results: axonal retrograde transport in TgSOD1 G93A was delayed compared to wild-type and Dync1h1 Loa/+ littermates, whereas it was faster in the TgSOD1 G93A –Dync1h1 Loa/+ double mutants [16] . This result may indicate an unexpected interaction, either direct or indirect, between Dync1h1 and mutant (and possibly wild-type) SOD1 [10,34] .

The extension of lifespan in TgSOD1 G93A –Dync1h1 Loa/+ double mutants was also shown by two other groups: Chen, Popko, and colleagues, who found a 21% increase in the lifespan of these mice compared to TgSOD1 G93A littermates [3] and Ilieva, Cleveland, and colleagues, who reported a 9% increase [14] . The latter group crossed their Dync1h1 Loa/+ mice with two other transgenic animals with different mutations in SOD1 that also give models of ALS they found no ameliorating effect of the dynein subunit mutation, which they suggest may reflect a difference in SOD1 protein levels in the transgenics.

Interestingly, Teuchert and colleagues reported that TgSOD1 G93A –Dync1h1 Cra1/+ double mutants also have a delayed disease onset compared to TgSOD1 G93A littermates [26] , but this effect is not seen in TgSOD1 G93A –Dync1h1 Swl/+ double mutants [3] , indicating that different members of this dynein subunit allelic series may well have different interactions. This also highlights the use of having an allelic series of mutants for helping to tease out protein–protein interactions that take place in different domains.

New research indicates that the role of the dynein HC mutation in ameliorating the effect of the TgSOD1 G93A transgene may lie in a surprising alteration in mitochondrial function that is found in the Dync1h1 Loa/+ and TgSOD1 G93A –Dync1h1 Loa/+ double mutant mice [10] . Morsi El-Kadi, Hafezparast, and colleagues have shown that in the double mutants the Dync1h1 Loa mutation leads to a significant reduction in the amount of toxic SOD1 G93A protein in the mitochondrial matrix, resulting in amelioration of the defects in mitochondrial membrane potential and respiration. The precise mechanism by which mutant dynein affects the differential deposition of SOD1 G93A in the double mutants and its role in ameliorating mitochondrial function is not understood yet. But, taking into account the role of dynein in mitochondrial transport and possibly mitochondrial fission [30] , it is likely that the Dync1h1 Loa mutation improves the transport of mitochondria and that the improved transport could make them less prone to mutant SOD1 association, leading to ameliorated membrane potential and respiration and/or reducing the association of mutant SOD1 protein with the mitochondrial membranes via an as-yet-unknown mechanism thus, the Dync1h1 Loa mutation could restore the docking of dynein (and kinesin) to the mitochondria to improve its transport ( Fig. 18.6 ) [10,29] .

Figure 18.6 . Effects of the Dync1h1 Loa mutation on the TgSOD1 G93A mouse phenotype. mSOD1, mutant SOD1 ●, mSOD1 aggregates ?, not tested yet.

The unexpected outcome of crossing the Dync1h1 Loa/+ and TgSOD1 G93A mice has prompted other crosses with the dynein HC subunit mutants. Ravikumar, Acevedo-Arozena, Rubinsztein, and colleagues crossed the Dync1h1 Loa/+ mouse with a mouse model of the neurodegenerative disorder Huntington’s disease (Hdh HD/+ ) [23] . They found that the Huntington’s phenotype, including tremor onset, motor coordination, and muscle function, was enhanced in compound heterozygote animals. This effect likely resulted from effects of the dynein mutation on the autosome–lysosome fusion process – showing a potential novel role for the dynein HC subunit in this pathway.

In other mouse models of neurological disorders, the Dync11h Loa/+ has had no effect on phenotype [2] .

Additional files

Additional file 1:

Sequences of each gene and CRISPR/Cas9-induced mutants or synthesized templates used in all experiments.

Additional file 2: Table S1.

List of primers used in this study.

Additional file 3: Fig. 1.

The yield of temperature gradient PCR with different single point mutation templates of NtCRTISO (synthesized) and different combination of primers was detected by agarose gel electrophoresis.

Additional file 4: Fig. 2.

The yield of temperature gradient PCR with different multiple point mutation templates of NtCRTISO (synthesized) and different combination of primers was detected by agarose gel electrophoresis.

Additional file 5: Fig. 3.

Identification of CRISPR/Cas9-induced crtiso mutants in tobacco by MSBSP-PCR.

Additional file 6: Fig. 4.

The sequencing and sequences analysis of different clones of NtCRTISO transgenic lines.

Additional file 7: Fig. 5.

Identification of CRISPR/Cas9-induced myb86 mutants in tobacco by MSBSP-PCR.

Additional file 8: Fig. 6.

The sequencing and sequences analysis of different transgenic lines of NtMYB86.

Additional file 9: Fig. 7.

Identification of CRISPR/Cas9-induced ggpps1 mutants in tobacco by MSBSP-PCR.

Additional file 10: Fig. 8.

The sequencing and sequences analysis of different transgenic lines of NtGGPPS1.

Additional file 11: Fig. 9.

Identification of CRISPR/Cas9-induced rin4 mutants in tobacco by MSBSP-PCR.

Additional file 12: Fig. 10.

The sequencing and sequences analysis of different transgenic lines of NtRIN4.

Additional file 13: Fig. 11.

Identification of CRISPR/Cas9-induced pvy mutants in tobacco by MSBSP-PCR.

Additional file 14: Fig. 12.

The sequencing and sequences analysis of different transgenic lines of NtPVY.

Watch the video: Gene mutation for genetic variability (December 2021).