ライフサイエンスコーパス: ribozyme

1 vidual RNA molecule with catalytic activity (ribozyme).

2 es that were replicated by an RNA polymerase ribozyme.

3 e the structure and catalytic mechanism of a ribozyme.

4 structure of a 2'-OCH3 -U5 modified twister ribozyme.

5 for destabilizing mutations in the Azoarcus ribozyme.

6 ectivity prevent the complete folding of the ribozyme.

7 ind RNA-puzzle challenge, the lariat-capping ribozyme.

8 -acting C75U-inhibited structures of the HDV ribozyme.

9 e was proportional to the fraction of active ribozyme.

10 ers, as well as catalytic bond scission in a ribozyme.

11 any naturally occurring small self-cleaving ribozyme.

12 catalysis of a model RNA enzyme, the hairpin ribozyme.

13 creases the ATPase stimulation by the folded ribozyme.

14 n the folding kinetics of the Diels-Alderase ribozyme.

15 nsights into the mechanism of this universal ribozyme.

16 n on the folding and function of the hairpin ribozyme.

17 ate, docked into the catalytic domain of the ribozyme.

18 3' end produced by self-cleavage of a delta ribozyme.

19 the poor turnover efficiency of the twister ribozyme.

20 re-catalytic structure of the twister-sister ribozyme.

21 ound previously also for the related twister ribozyme.

22 ays soft, non-specific interactions with the ribozyme.

23 inase ribozyme, making this a first-in-class ribozyme.

24 -way junctional twister-sister self-cleaving ribozyme.

25 actorial origins of catalysis by the twister ribozyme.

26 ubdomain of the 'Tetrahymena' group I intron ribozyme.

27 rtiary free-energy landscape of the Azoarcus ribozyme.

28 serving as unfolded templates and effective ribozymes.

29 used to both analyze and engineer allosteric ribozymes.

30 g a size comparable to that of large natural ribozymes.

31 experimental validation of novel functional ribozymes.

32 strategies proposed for small self-cleaving ribozymes.

33 ent the evolution of functional RNAs such as ribozymes.

34 to the catalytic mechanism of lariat-forming ribozymes.

35 up I introns, the Twort and Azoarcus group I ribozymes.

36 ring in active sites of nuclease enzymes and ribozymes.

37 ularly of long, structured sequences such as ribozymes.

38 NA molecules including rRNA, tRNA, snRNA and ribozymes.

39 ustain a genome long enough to encode active ribozymes.

40 tic strategies employed by small nucleolytic ribozymes.

41 of the 5'-exon) catalyzed by group II intron ribozymes.

42 tely the same rate constant as the wild-type ribozyme (~1 min(-1)).

43 valuation as catalytic cofactors of the glmS ribozyme, a bacterial gene-regulatory RNA that controls

44 al structures available for all of the known ribozymes, a major challenge involves relating functiona

45 The hairpin ribozyme accelerates a phosphoryl transfer reaction with

46 th, the resulting internal dilution produced ribozyme activation.

47 ys an important role in the stabilization of ribozyme active conformations in vivo.

48 A model of the ribozyme active site is proposed that accommodates these

49 teraction, is sufficient for stabilizing the ribozyme active site, including alignment of the attacki

50 er free Mg(2+) concentrations decrease their ribozyme activity and constitute a natural barrier to gr

51 stabilized to Mg(2+), which is required for ribozyme activity and RNA synthesis.

52 this network of tertiary interactions reduce ribozyme activity in physiological Mg(2+) concentrations

53 tatic behaviour: the maintenance of constant ribozyme activity per unit volume during protocell volum

54 onucleotides within fatty acid vesicles, and ribozyme activity was inhibited.

55 h specific and random sequence, can modulate ribozyme activity.

56 iFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a

57 bilize the structure of the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiolog

58 f matR expression via synthetically designed ribozymes altered the processing of various introns, inc

59 However, we recently observed that both a ribozyme and an RNA aptamer retain considerable function

60 integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to eng

61 dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to

62 that molecular crowders stabilize the native ribozyme and favor the active structure relative to comp

63 s, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles

64 tion is required for complete folding of the ribozyme and stabilization of the active site.

65 des resulting from the 3' end created by the ribozyme and the 5' end created from an endolytic cleava

66 lations to investigate the mechanism of this ribozyme and to elucidate the roles of the catalytic met

67 folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep seq

68 assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate.

69 two constructs, an exact monomer flanked by ribozymes and a trihelix-forming RNA with requisite 5' a

70 es, which enables better characterization of ribozymes and aptamers.

71 nalysis efficiently identified highly active ribozymes and estimated catalytic activity with good acc

72 For ribozymes and riboswitches, the RNA structure itself pro

73 obust technologies developed for visualizing ribozymes and riboswitches, together with new approaches

74 ate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleava

75 been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been ex

76 nucleic acid (SNA) architecture to stabilize ribozymes and transfect them into live cells is reported

77 the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute es

78 nally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymeras

79 hat multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed

80 nsist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase,

81 rans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited stru

82 stabilization of the transition state by the ribozyme, and functional group substitution at G33 indic

83 t the accessible conformational space of the ribozyme, and that these so-called topological constrain

84 complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA.

85 How then could self-replicating ribozymes appear, for which recent experiments suggest a

86 siRNAs, ribozymes, aptamers, chemical ligands, fluorophores and

87 that large hepatitis delta virus (HDV)-like ribozymes are activated by peripheral domains that bring

88 Self-cleaving ribozymes are found in all domains of life and are belie

89 Ribozymes are highly structured RNA sequences that can b

90 intron recognition duplex of a self-splicing ribozyme as a model system to study the influence of Mg(

91 tertiary structure formation of the hairpin ribozyme as a model to probe the effects of polyethylene

92 Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applie

93 design of fast-cleaving engineered synthetic ribozymes as RNA nucleolytic reagents and as subjects fo

94 olecular dynamics simulations of the hairpin ribozyme at different stages along the catalytic pathway

95 and free energy calculations of the twister ribozyme at different stages along the reaction path to

96 ng and helix assembly of a bacterial group I ribozyme at different temperatures and in different MgCl

97 ice, our work supports the implementation of ribozyme-based devices in applications requiring the det

98 itro cleavage rate constants associated with ribozyme-based devices is essential for advancing the mo

99 Here, we develop ribozyme-based devices that respond to protein ligands i

100 nce our ability to characterize and engineer ribozyme-based devices.

101 , four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacteri

102 lements that regulate the activity of the LC ribozyme by conformational switching and suggest a mecha

103 activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize funct

104 a three-way junction variant of the hairpin ribozyme can be stabilized by specific insertion of a sh

105 The reverse transcriptase ribozyme can incorporate all four dNTPs and can generate

106 One mechanism by which ribozymes can accelerate biological reactions is by adop

107 Here we show that RNA polymerase ribozymes can assemble from simple catalytic networks of

108 Ribozymes can catalyze phosphoryl or nucleotidyl transfe

109 This study showed that ribozymes can use trimetaphosphate to triphosphorylate R

110 res enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of

111 Ribozyme-catalysed nucleosidation-the key biosynthetic s

112 a replicating protocell with an RNA genome, ribozyme-catalysed peptide synthesis might have been suf

113 Nucleolytic ribozymes catalyze site-specific cleavage of their phosp

114 Group II intron ribozymes catalyze the cleavage of (and their reinsertio

115 he canonical RNA world in which RNA enzymes (ribozymes) catalyze replication of the RNA genomes of pr

116 ics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.

117 at can reveal deep mechanistic insights into ribozyme-catalyzed reactions.

118 the origin of life prior to the evolution of ribozyme-catalyzed RNA replication.

119 The glmS ribozyme catalyzes a self-cleavage reaction at the phosp

120 The hepatitis delta virus (HDV) ribozyme catalyzes a self-cleavage reaction using a comb

121 The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a cata

122 econd thiophosphorylation, implying that the ribozyme catalyzes both phosphoryl and nucleotidyl trans

123 ctural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reactio

124 The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligati

125 One of the key challenges encountered in ribozyme characterization is the efficient generation of

126 We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonan

127 ments is found to result in the emergence of ribozyme cleavage function, thus establishing a connecti

128 Similarities to the hairpin ribozyme cleavage loop activation suggest general strate

129 Importantly, the ribozyme cleavage reaction of the emissive fluorescent t

130 ioreactors is established by demonstrating a ribozyme cleavage reaction within the liposome-coated dr

131 ere conditions generally lead to significant ribozyme cleavage.

132 h allows continuous, real-time monitoring of ribozyme cleavage.

133 pproximately 70-fold increase in the rate of ribozyme cleavage.

134 egies to enhance fidelity in RNA folding and ribozyme cleavage.

135 g indicates that ATPase activity tracks with ribozyme compactness.

136 Endonucleolytic ribozymes constitute a class of non-coding RNAs that cat

137 Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site

138 viously thought; the catalytic repertoire of ribozymes continues to expand, approaching the goal of s

139 ides are brought into close proximity at the ribozyme core through long-range interactions mediated b

140 n a linked transition and assembles with the ribozyme core via three tertiary contacts: a kissing loo

141 g arises from a topological error within the ribozyme core, and a specific topology is proposed.

142 talysis of bond scission in these hammerhead ribozymes could be restored by putative t2M/t4M refoldin

143 By use of these allosteric ribozymes, cytoplasmic concentrations of c-di-GMP in thr

144 unctional switches in a family of hammerhead ribozymes deactivated by stem or loop replacement with a

145 d a 1.55-A crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditio

146 ous types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtain

147 Ribonuclease P (RNase P) is one of the first ribozymes discovered and it is found in all phylogenetic

148 helices in diverse structured RNAs including ribozyme domains, riboswitch aptamers, and viral RNA dom

149 ve-site Mg(2+) cation to N7 of G33 makes the ribozyme drastically slower.

150 the P4-P6 domain of the Tetrahymena group I ribozyme embedded in Xenopus egg extract demonstrate the

151 However, how such replicase ribozymes emerged from the pools of short RNA oligomers

152 Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy,

153 calable generation of functional full-length ribozyme-encoding RNA.

154 anges are required in the minimal hammerhead ribozyme enzyme strand sequence (providing that the natu

155 Azoarcus form spontaneously in the unfolded ribozyme even at very low Mg(2+) concentrations, and are

156 small-angle X-ray scattering showed that the ribozyme expands rapidly to intermediates from which P3

157 Most ribozyme families have distinct catalytic cores stabiliz

158 imates of catalytic activity for hundreds of ribozyme families.

159 imately giving rise to an original branching ribozyme family.

160 essful proof-of-principle use of multiplexed ribozyme flanked gRNAs to induce mutations in vivo in Dr

161 Self-cleaving ribozymes fold into intricate structures, which orient a

162 anonical RNA-binding proteins that stabilize ribozyme folding; the apparent chaperoning activity of t

163 The Tetrahymena group I intron ribozyme folds in vitro to a long-lived misfolded confor

164 uctured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediat

165 When Mg(2+) is replaced by Ca(2+) the ribozyme folds, but the active site remains unstable.

166 the conserved evolutionary mechanism used by ribozymes for catalysis.

167 platform can also be used to screen scissile ribozymes for improved catalysis.

168 e a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymeriza

169 ndividual structural elements of the group I ribozyme from the bacterium Azoarcus form spontaneously

170 of the large sequence space relevant to the ribozyme function.

171 ffect polymerase fidelity and participate in ribozyme general acid-base catalysis.

172 By synthesizing Azoarcus ribozyme genotypes that differ in their single-nucleotid

173 issing loop junction of the Varkud Satellite ribozyme has been experimentally characterized, the dyna

174 An RNA replicase ribozyme has long been sought by chemists interested in

175 Neither in splicing nor for self-cleaving ribozymes has the role of the two Mg(2+) ions been estab

176 Artificial ribozymes have been designed in the past either manually

177 Small self-cleaving ribozymes have been discovered in all evolutionary domai

178 Hammerhead ribozymes have been observed to be active in the presenc

179 Additionally, self-cleaving ribozymes have been the subject of extensive engineering

180 ons, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40.

181 t structural features inherited from group I ribozymes have undergone speciation due to profound chan

182 recent crystal structure of the precleavage ribozyme identified a Mg(2)(+) ion that interacts throug

183 ndicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary fol

184 al model for the active state of the twister ribozyme in solution that is consistent with these and o

185 e the structure of the inactive state of the ribozyme in the absence of magnesium.

186 S measurements on a 64 kDa bacterial group I ribozyme in the presence of mono- and divalent ions and

187 The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for pepti

188 ife, we evolved populations of self-cleaving ribozymes in an anoxic atmosphere with varying pH in the

189 mple genetic system, an evolving RNA enzyme (ribozyme) in which a combination of high throughput geno

190 he docked, catalytically active state of the ribozyme, in part through excluded volume effects; unexp

191 n other ribozymes such as the hairpin and VS ribozymes, in the twister ribozyme there may be a twist.

192 The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic

193 The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for pro

194 The improved polymerase ribozyme is able to synthesize a variety of complex stru

195 ructure of the in-line aligned env22 twister ribozyme is compared with two recently reported twister

196 A minimal version of the twister ribozyme is reported that lacks the phylogenetically con

197 The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of t

198 ficiency of CYT-19-mediated unfolding of the ribozyme is tightly linked to global RNA tertiary stabil

199 he lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms.

200 We find that kinase ribozyme K28(1-77)C, in contrast with previously charact

201 s of ribose hydroxyls to catalysis by kinase ribozyme K28.

202 However, existing polymerase ribozymes lack the capacity to synthesize RNAs approachi

203 er has previously been reported for a kinase ribozyme, making this a first-in-class ribozyme.

204 at cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ens

205 Base-modifying ribozymes may have played important roles in early RNA w

206 embranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other

207 ey positions, a mechanistic insight into the ribozyme-mediated cleavage is gained.

208 ar functions, and are integral components of ribozymes, mRNA, and riboswitches.

209 Structured RNAs such as ribozymes must fold into specific 3D structures to carry

210 This compensation helps explains why ribozyme mutations are often less deleterious in the cel

211 covery of a new class of small self-cleaving ribozymes named Pistol.

212 otif represents a new class of self-cleaving ribozymes of yet unknown biological function.

213 tertiary contacts of the Tetrahymena group I ribozyme on the dynamics of its substrate helix (referre

214 ivity were measured for wild type and mutant ribozymes over a range of conditions.

215 nditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condi

216 obtained a complete activity profile of the ribozyme pool which was used to both analyze and enginee

217 eases the genotypic diversity of an evolving ribozyme population.

218 resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombinati

219 by some endoribonucleases and self-cleaving ribozymes produces RNA fragments with 5'-hydroxyl (5'-OH

220 lf-splicing introns, long proposed to be the ribozyme progenitor of spliceosome.

221 vity of CYT-19, suggesting that destabilized ribozymes provide more productive interaction opportunit

222 cleophile for the required inline hammerhead ribozyme reaction mechanism.

223 ontrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thio

224 he substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate

225 hese structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication.

226 th ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric pep

227 led observations of Na(+) ion binding in the ribozyme's active site.

228 presence of the crowding agent show that the ribozyme's activity increases while the heterogeneity de

229 The protocol is analogous to (deoxy)ribozyme selections, but it enables the development of f

230 Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine b

231 tion efforts that either activate or inhibit ribozyme self-cleavage upon ligand binding to the aptame

232 ic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG),

233 The properties of this novel ribozyme-SNA are characterized in the context of the tar

234 es such as PEG stabilize a bacterial group I ribozyme so that the RNA folds in low Mg(2+) concentrati

235 dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth.

236 m of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures

237 in previously observed variations in hairpin ribozyme stability.

238 rom what is currently the highest-resolution ribozyme structure in the Protein Data Bank.

239 Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutio

240 compared with two recently reported twister ribozymes structures, which adopt similar global folds,

241 acid acting through the N1 position in other ribozymes such as the hairpin and VS ribozymes, in the t

242 nd phosphate mutations in the twister-sister ribozyme, suggest contributions to the cleavage chemistr

243 d relative to the profiles for the wild-type ribozyme, suggesting that the A25.C20 double mutant has

244 ion studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucl

245 ting aptamers that exhibit ligand-responsive ribozyme tertiary interactions.

246 The correct folding of the active site and ribozyme tertiary structure is also regulated by metal i

247 ture as the medium has led to discovery of a ribozyme that can catalyse polymerization of an RNA chai

248 ter RNA is a recently discovered nucleolytic ribozyme that is present in both bacteria and eukarya.

249 e strongest ATPase stimulation occurs with a ribozyme that lacks all five tertiary contacts and does

250 We report a single ribozyme that performs both reactions, with a nucleobase

251 rt a 3.3 A crystal structure of the complete ribozyme that reveals the active, shifted conformation o

252 study an artificial 2'-5' AG1 lariat-forming ribozyme that shares the sequence specificity of lariat

253 Group II introns are Mg(2+)-dependent ribozymes that are considered to be the evolutionary anc

254 Group II introns are large, autocatalytic ribozymes that catalyze RNA splicing and retrotransposit

255 an in vitro selection strategy for isolating ribozymes that catalyze the triphosphorylation of RNA 5'

256 es are small, ligand-dependent self-cleaving ribozymes that function independently of transcription f

257 Further, unlike ribozymes that motivated the RNA World hypothesis, Class

258 been reported of a new catalytic RNA, the TS ribozyme, that has been identified through comparative g

259 two-piece version of the Tetrahymena group I ribozyme, the separated P5abc subdomain folds to local n

260 tat, and (f) replication mediated in some by ribozymes, the fingerprint of the RNA world.

261 rotein complexes, the nature of catalysis by ribozymes, the structural basis of recognition of RNA by

262 the hairpin and VS ribozymes, in the twister ribozyme there may be a twist.

263 the case of the hepatitis delta virus (HDV) ribozyme, there are three high-resolution crystal struct

264 We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p's (2',3'-c

265 linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we

266 The unexpected ability of an RNA polymerase ribozyme to copy RNA into DNA has ramifications for unde

267 ct mutants of the Tetrahymena group I intron ribozyme to demonstrate that the efficiency of CYT-19-me

268 the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiological Mg(2+) concentrati

269 he promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA.

270 After allowing the ribozyme to radiolabel itself by phosphoryl transfer fro

271 o nanoparticles to deliver siRNA, miRNA, and ribozymes to cancer and virus-infected cells.

272 The chemical strategies used by ribozymes to enhance reaction rates are revealed in part

273 r molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg(2+) concentrations

274 airing interaction in the minimal hammerhead ribozyme transforms an RNA sequence possessing typically

275 red from structure, and suggest that the HDV ribozyme transition state resembles the cleavage product

276 tabilized the active structure of the mutant ribozymes under physiological conditions.

277 Our results suggest that group I ribozymes utilize the same interactions with specific me

278 tative characterization of greater than 1000 ribozyme variants in a single experiment.

279 Efficient unfolding of destabilized ribozyme variants is accompanied by increased ATPase act

280 We generated a library of predefined ribozyme variants that were converted to DNA and analyze

281 h concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining ne

282 f P4-P6, a domain of the Tetrahymena group I ribozyme, via single-molecule fluorescence resonance ene

283 eral active sequences were isolated, and one ribozyme was analyzed in more detail.

284 odel system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 r

285 A highly evolved RNA polymerase ribozyme was found to also be capable of functioning as

286 The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of u

287 The ribozyme was truncated to 96 nt, while retaining full ac

288 atalytic site of a minimal type I hammerhead ribozyme were replaced with oligo-U loops, severely crip

289 rinting polymers, peptide nucleic acids, and ribozymes were encompassed as "products" of biomimetic c

290 ness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evol

291 cribing the Mg(2+)-mediated folding of these ribozymes were previously determined by time-resolved hy

292 es increased the biochemical activity of the ribozyme, whereas sucrose did not.

293 2.9-A crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilize

294 inach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI)

295 eled adenine at this position in the twister ribozyme, which is significantly shifted compared to the

296 Ribozymes, which carry out phosphoryl-transfer reactions

297 tures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inheri

298 anscripts were used to assemble a hammerhead ribozyme with all permutations of natural and modified e

299 Kinetic analysis of ribozymes with systematically altered length and stabili

300 le-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-base