ライフサイエンスコーパス: sgRNA

1 sgRNA expression cassettes were stably integrated into t

2 erived vector (TRBO) designed with 5' and 3' sgRNA proximal nucleotide-processing capabilities.

3 m 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small num

4 th minimal false positives using a compact 5 sgRNA/gene library.

5 In this work, we describe a novel 5' sgRNA of BMV (sgRNA3a) that we propose arises by prematu

6 Cas9 cleavage assays only edited DNA when 5' sgRNA nucleotide overhangs were removed, suggesting a no

7 RNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a diseas

8 However, we were able to observe a sgRNA produced from the native 5'TR of a small defective

9 ng asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of t

10 ct binding of RNA-2 to RNA-1 trans-activates sgRNA synthesis.

11 omated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking.

12 ession of an RNA transcript consisting of an sgRNA adjoining a GFP protein coding region produced ind

13 smallest Cas9 orthologs, in complex with an sgRNA and its target DNA.

14 single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primar

15 ovides more reliable off-target analyses and sgRNA design.

16 prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularl

17 ered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injectio

18 RNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency.

19 nsive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest

20 Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing st

21 that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generati

22 Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacil

23 rolled codelivery of intact Cas9 protein and sgRNA.

24 ined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressi

25 rand RNA3 as a template for genomic RNA3 and sgRNA syntheses.

26 ethylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modificatio

27 ferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT

28 results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-me

29 ually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse g

30 covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/d

31 lls can be determined by competition between sgRNA and intracellular RNA molecules for the binding to

32 he recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and Ru

33 Blocking sgRNA transcription stimulated RNA3 replication by up to

34 l three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 acc

35 machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyo

36 as used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence pro

37 The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and te

38 Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each c

39 The Cas9-sgRNA complex binds to DNA elements complementary to the

40 earching and targeting mechanism of the Cas9-sgRNA complex, investigating chromosome organization, an

41 Guided by the structure of the Cas9-sgRNA complex, we identify regions of sgRNA that can be

42 unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic lo

43 Efficient genome editing with Cas9-sgRNA in vivo has required the use of viral delivery sys

44 ons that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, r

45 DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site.

46 ectroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% ef

47 iple for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment m

48 Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic

49 occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencie

50 these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice

51 Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the i

52 urthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several

53 Moreover, we observed the Cas9/sgRNA complex bound to the DNA substrates and characteri

54 mpetitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecti

55 rges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target seque

56 h a pulse exposure of the genome to the Cas9/sgRNA complex.

57 NA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function.

58 The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust t

59 Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well

60 leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and

61 ious organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single clea

62 d vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nucle

63 Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the ba

64 elivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured h

65 ficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achiev

66 as9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in me

67 HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot num

68 Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modificatio

69 At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for ant

70 mosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISP

71 We generated complex sgRNA libraries with unique molecular identifiers (UMIs)

72 implies that the 3'-terminal segment confers sgRNA the ability to withstand competition from non-spec

73 nstructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential gene

74 triction enzyme to derive a densely covering sgRNA library from input DNA.

75 ation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulte

76 for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.

77 SFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci

78 The dCas9/sgRNA binary complex is stable and binds its target DNA

79 ur results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a

80 ce the Cas9/TRBO-sgRNA platform demonstrated sgRNA flexibility, we targeted the N. benthamiana NbAGO1

81 'TR closer to the 3' terminus, demonstrating sgRNA promoter activity of the native 5'TR.

82 ular Chipper technology for generating dense sgRNA libraries for genomic regions of interest, and a p

83 Here, we use recently devised sgRNA design rules to create human and mouse genome-wide

84 '-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the m

85 y published screens performed with different sgRNA libraries.

86 stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-targe

87 When using a dual sgRNA system, we achieved complete PDX1 disruption.

88 We found that "dual-sgRNA targeting" is essential for biallelic knockin of F

89 cle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single int

90 Each sgRNA is efficiently expressed and can mediate multiplex

91 ORF) is believed to be transcribed from each sgRNA.

92 majority of published "rules" for efficient sgRNA design do not effectively predict germline transmi

93 d ORF-specific primers identified only eight sgRNA species.

94 diac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh

95 equences upstream of the start site enhanced sgRNA promoter activity.

96 ctation-maximization to iteratively estimate sgRNA knockout efficiency and gene essentiality.

97 creased false-positive results and estimated sgRNA activity for both this data set and previously pub

98 screens, we compare our approach to existing sgRNA design and expression strategies.

99 KO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic scree

100 For sgRNA transcription, viral-based TRV and synthetic binar

101 Current methods for sgRNA design are mainly concerned with predicting off-ta

102 we proposed an accurate prediction model for sgRNA design efficiency.

103 ick chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as

104 The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout effici

105 Furthermore, sgRNA targeting GPI anchor protein pathway genes induced

106 nts in noncoding regions requires generating sgRNA libraries that are densely covering, and ideally i

107 rned with predicting off-targets for a given sgRNA using basic sequence features and employ elementar

108 y of a genomic site to be cleaved by a given sgRNA.

109 However, sgRNA's vary widely in their activity and models for pre

110 redictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens.

111 acilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.

112 e enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA

113 l candidates were validated using individual sgRNA and complementary DNA overexpression.

114 alyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo.

115 CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates

116 intly measure a cell's transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-

117 nding activity of SaCas9 and to optimize its sgRNA scaffold.

118 of extraneous nucleotides, which has limited sgRNA expression by delivery vectors.

119 ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 %

120 The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimeri

121 t here a set of pre-designed human and mouse sgRNA sequences that are optimized for both high on-targ

122 nomic RNA amplification and subgenomic mRNA (sgRNA) transcription.

123 g, translational frameshifting, and multiple sgRNA formation, but phylogenetically the CTV polymerase

124 In addition, we have developed a multiplexed sgRNA expression strategy that promotes the functional a

125 ssential part of the genome from which a new sgRNA was expressed.

126 cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese B

127 Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in vari

128 rmine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target

129 ere, we increased cellular concentrations of sgRNA by transiently delivering sgRNAs using a Tobacco m

130 (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely

131 s9 platform is conferred through the ease of sgRNA programmability as well as the degree of modificat

132 , to determine target-specific efficiency of sgRNA.

133 echanical analysis for the whole ensemble of sgRNA-target complex conformations, we identify a strong

134 By a systematic investigation of sgRNA structure we find that extending the duplex by app

135 s to be a regulatory mechanism for levels of sgRNA production.

136 shown to be useful in defining mechanisms of sgRNA synthesis.

137 were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing

138 n in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in

139 This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by co

140 e Cas9-sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancin

141 f viral gene expression is the regulation of sgRNA synthesis by specific promoter elements.

142 hensive computational tool based on a set of sgRNA design rules summarized from these published repor

143 C percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage effi

144 is shown to be required for transcription of sgRNA.

145 The number of sites identified depended on sgRNA sequence and nuclease concentration.

146 the N. benthamiana NbAGO1 paralogs with one sgRNA and also multiplexed two sgRNAs using a single TRB

147 putational design rules and create optimized sgRNA libraries that maximize on-target activity and min

148 In addition, the optimized sgRNA structure also significantly increases the efficie

149 Optimizing sgRNA design to improve the efficiency of target/DNA cle

150 enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of

151 r distinct PAM specificities and orthologous sgRNA recognition.

152 rkflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be

153 es a computational sequence model to predict sgRNA efficiency, and employs a specificity scoring func

154 derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments.

155 ckout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments.

156 is of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively ca

157 nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation.

158 sgRNA) and its genomic target, which refines sgRNA design.

159 y to simultaneously assess single guide RNA (sgRNA) activity across approximately 1,400 genomic loci.

160 f SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing t

161 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA desig

162 nes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein archite

163 ring tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing.

164 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency v

165 Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce h

166 In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA s

167 eatures that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens.

168 n (Cas9) and an engineered single guide RNA (sgRNA) genome editing platform that offers revolutionary

169 enes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome edi

170 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs)

171 nce from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered.

172 ection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one

173 xture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes.

174 ivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn-like DNA nanopart

175 Single-guide RNA (sgRNA) is one of the two key components of the clustered

176 ning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method

177 a genome-scale lentiviral single-guide RNA (sgRNA) library.

178 simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISP

179 The single guide RNA (sgRNA) of the system recognizes its target sequence in t

180 9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins.

181 We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identi

182 et selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the targ

183 ified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targ

184 n complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional act

185 sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently dir

186 ple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites o

187 Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardio

188 ic Repeats system allows a single guide RNA (sgRNA) to direct a protein with combined helicase and nu

189 9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity.

190 s9 system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 to virtually anyw

191 9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there a

192 The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA ef

193 ence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that m

194 tering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites

195 ein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of rep

196 cture at the 5' end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcrip

197 matics tools for design of single guide RNA (sgRNA), which determines the efficacy and specificity of

198 donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 c

199 sequence of an associated single guide RNA (sgRNA).

200 an asRNA that sequesters a small guide RNA (sgRNA).

201 or to an aptamer-modified single guide RNA (sgRNA).

202 protein and a customizable single guide RNA (sgRNA).

203 plementary to a programmed single guide RNA (sgRNA).

204 protein in complex with a single guide RNA (sgRNA).

205 ased genetic screens using single-guide-RNA (sgRNA) libraries have proven powerful to identify geneti

206 protein is translated from a subgenomic RNA (sgRNA) that is transcribed from genomic RNA-1.

207 l relies on well-designed single guide RNAs (sgRNA).

208 w, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permi

209 Several sgRNA design tools have been developed for gene editing,

210 low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four

211 A single sgRNA can induce small insertions or deletions that part

212 While the use of a single sgRNA was efficient at inducing mutated fetuses, the lac

213 transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that r

214 Embryos microinjected with single sgRNA targeting FOXN1, RAG2, IL2RG or PRKDC were pooled

215 advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition a

216 molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg(2+) bound.

217 However, the efficacy of a specific sgRNA is not uniquely defined by exact sequence homology

218 ntinued to produce a 5'-terminal plus-strand sgRNA, here much larger ( approximately 11 kb), apparent

219 Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding posi

220 ility of the corresponding positive-stranded sgRNA as a messenger.

221 monstrating that neither 5'- nor 3'-terminal sgRNA is necessary for replication of the replicon or fu

222 A large 3'-terminal sgRNA resulting from the putative promoter activity of t

223 Surprisingly, a new 3'-terminal sgRNA was observed from the duplicated 5'TR.

224 T1 RNAs and presumably the large 3'-terminal sgRNA while having no impact on replication, demonstrati

225 Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1

226 icity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potent

227 We further found that sgRNA transcription inhibited RNA3 replication at a step

228 NA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication.

229 Last, we show that sgRNA efficiency is associated with specific sequence mo

230 Our data suggest that sgRNA 2 may be bicistronic, expressing both ORF 2a and O

231 nition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at

232 cient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offer

233 indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Ca

234 In addition to this targeting function, the sgRNA has also been shown to play a role in activating t

235 es, particularly when the mismatch is in the sgRNA "seed" region.

236 terized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM).

237 the BYDV genome containing mutations in the sgRNA promoter.

238 corporating MS2 or PP7 RNA aptamers into the sgRNA.

239 y as well as the degree of modifications the sgRNA can tolerate without compromising its association

240 out efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutat

241 h PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target effici

242 rties and recent engineering advances of the sgRNA component in Cas9-mediated genome targeting.

243 ut is highly dependent on the potency of the sgRNA guide sequence.

244 importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are

245 mutation, particularly if the potency of the sgRNA is also low.

246 This dual function of the sgRNA likely underlies observations that different sgRNA

247 tending the tetraloop and stem loop 2 of the sgRNA with MS2 or PP7 aptamers enhances the signal-to-ba

248 ltering the hybridization free energy of the sgRNA-asRNA complex.

249 Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis.

250 ucleotide genomic match at the 5' end of the sgRNA.

251 novel antisense transcript downstream of the sgRNA/dCas9-binding site.

252 Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engin

253 r PP7 aptamers to different locations on the sgRNA, we found that extending the tetraloop and stem lo

254 approximately 10 to >1,000 depending on the sgRNA.

255 the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitu

256 The data obtained suggest that the sgRNA transcription of CTV is dissimilar from the corona

257 DNA strand that is not complementary to the sgRNA (nontarget strand).

258 x binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript el

259 Consistent with this, sgRNA transcription was stimulated by up to 400% when mu

260 aled little or no homology between the three sgRNA promoter elements.

261 llus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary

262 Since the Cas9/TRBO-sgRNA platform demonstrated sgRNA flexibility, we target

263 0% within 7 d postinoculation using the TRBO-sgRNA constructs, which retained 5' nucleotide overhangs

264 nstrate proof-of-principle, we used the TRBO-sgRNA delivery platform to target GFP in Nicotiana benth

265 We also demonstrate that the tRNA-sgRNA system markedly increases the efficacy of conditio

266 The current commonly used sgRNA structure has a shortened duplex compared with the

267 aluate the sequence composition of the whole sgRNA and its surrounding region using models compiled f

268 We report a genome-wide sgRNA design tool and provide an online website for pred

269 Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A resolution.

270 rcially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mut