ライフサイエンスコーパス: sgRNA

1 Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted inte

2 of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9.

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

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

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

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

7 virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana bent

8 ibonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that

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

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

11 RNA (sgRNA) design tools mainly depend on a sgRNA sequence and the local information of the targeted

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

13 (RNP) complex between a Cas9 nuclease and an sgRNA.

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

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

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

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

18 ic interactions between ABP and aptamer, and sgRNA and Cas9 protein.

19 (inducing for nitA) were tested for Cas9 and sgRNA expression, and for the ability to generate progen

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

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

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

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

24 bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the

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

26 t the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA-O16B knocks out green fluorescent prot

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

28 y wild-type strain with the Cas9 plasmid and sgRNA plasmids targeting regA or invA yielded regA and i

29 rolled codelivery of intact Cas9 protein and sgRNA.

30 V) as a packaging vector for both SaCas9 and sgRNA.

31 cLNPs deliver siRNA and sgRNA to T cells at doses as low as 0.5 mg kg(-1) and, u

32 active state through the effects of another sgRNA.

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

34 set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice pro

35 by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes.

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

37 r cells that had been transduced with a Bcor sgRNA developed pro-B1 ALL, characterized by a B-1 proge

38 pro-B1 ALL cell lines established from Bcor sgRNA/NP23 recipients at clinically achievable concentra

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

40 c variations that are not distinguishable by sgRNA designing tools based on one reference genome.

41 have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioort

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

43 CLC, we performed a genome-scale CRISPR-Cas9 sgRNA screen.

44 w algorithm outperforms existing CRISPR/Cas9 sgRNA design tools.

45 From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylas

46 ngle, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker.

47 r genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic

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

49 alytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg(2+).

50 , and genome-editing machineries (e.g., Cas9-sgRNA ribonucleoprotein (RNP), and RNP together with don

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

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

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

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

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

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

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

58 t 5'-and 3'-nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo.

59 em, which allows efficient packaging of Cas9/sgRNA ribonucleoprotein (RNP).

60 as a powerful technology that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) to target and e

61 and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like particles via the specifi

62 cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo ado

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

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

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

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

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

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

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

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

71 Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a

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

73 TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple a

74 ese rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes esse

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

76 rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability.

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

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

79 Here, we develop a precisely controlled sgRNA expression cassette that can be combined with wide

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

81 ains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the h

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

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

84 o solve these issues, it is needed to design sgRNA with high cell-specific efficacy and specificity.

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

86 y published screens performed with different sgRNA libraries.

87 b-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows po

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

89 We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide do

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

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

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

93 ysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants.

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

95 to site-specific modifications that enhance sgRNA activity and in vivo stability.

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

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

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

99 tition assays with individual GFP-expressing sgRNA constructs.

100 ction of adeno-associated viruses expressing sgRNA-guided CjABE inhibited the growth of gliomas harbo

101 DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability.

102 eveloped Crisflash, a software tool for fast sgRNA design and potential off-target discovery, built f

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

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

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

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

107 Most currently available tools for sgRNA design operate only with standard reference genome

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

109 Furthermore, sgRNA targeting GPI anchor protein pathway genes induced

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

111 ays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of

112 promoter was used for expression of the GFP-sgRNA fusion transcript, it also produced indels when de

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

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

115 design, there is a pressing need for greater sgRNA potency and generalizability across various experi

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

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

118 ere have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA

119 rget effects caused by mismatch tolerance in sgRNA-DNA binding.

120 pegFinder can incorporate sgRNA on-target and off-target scoring predictions into

121 of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner and that e

122 smids encoding both SaCas9 and an individual sgRNA.

123 ry system with reduced recombination-induced sgRNA-barcode mispairing.

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

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

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

127 Coinclusion of a KDM5A sgRNA decreased SCLC tumorigenesis and metastasis, and t

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

129 ed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13

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

131 ngated progenitor sgRNAs, whereas the mature sgRNA end products are resistant.

132 sites and derived rules governing mismatched sgRNA activity using deep learning.

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

134 e-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that

135 tions of the guide RNA, and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like pa

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

137 ntravenous injection of BAMEA-O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocyte

138 ry of an improved Cas9 plasmid with multiple sgRNA plasmids and an efficient screening procedure to i

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

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

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

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

143 op a method for light-induced degradation of sgRNA termed CRISPRoff.

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

145 Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations

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

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

148 N facilitates controlled, rapid induction of sgRNA activity.

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

150 We build a library of sgRNA variants with different expression activation and

151 perimentally separating cellular mixtures of sgRNA from gRNA, is a unique advantage of our in-silico

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

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

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

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

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

157 bility of this method for quality testing of sgRNA, tRNA, and mRNA.

158 We show that replacing the Tetraloop of sgRNA scaffold with a com aptamer preserves the function

159 hrough precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary s

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

161 CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM

162 e find that the common practice of using one sgRNA can produce both unintended plasmid integration an

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

164 erns of shared genetic variation to optimize sgRNA design for different human populations.

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

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

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

168 iting systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitatio

169 scherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefi

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

171 r distinct PAM specificities and orthologous sgRNA recognition.

172 g the significance of spacing between paired sgRNA targets and the efficacy of NHEJ and HDR in repair

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

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

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

176 ely charged bridge helix, thereby preventing sgRNA loading.

177 of 11,776 genomically integrated protospacer-sgRNA pairs containing all possible NNNN PAMs.

178 ng an accurate mapping algorithm to quantify sgRNA levels, and minimizing the parameters that need to

179 ic regenerator (Reg) mutant strain receiving sgRNA plasmid with glsA protospacer sequence yielded pro

180 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon.

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

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

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

184 n mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex.

185 ne base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation.

186 elded from the active Cas9*single guide RNA (sgRNA) complex through the co-administration of dead-RNA

187 as9 gene editing dogma for single guide RNA (sgRNA) delivery is based on the premise that 5'-and 3'-n

188 Existing single-guide RNA (sgRNA) design tools mainly depend on a sgRNA sequence an

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

190 ozyme-guide-ribozyme (RGR) single guide RNA (sgRNA) expression strategy with RNA polymerase II promot

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

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

193 aging with high-throughput single guide RNA (sgRNA) identification in individual cells.

194 ion of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuti

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

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

197 ivery of Cas9 nuclease and single-guide RNA (sgRNA) into the specific cell and organ.

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

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

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

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

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

203 9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins.

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

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

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

207 for identifying CRISPR-Cas single guide RNA (sgRNA) targets.

208 ery of Cas9 nuclease and a single-guide RNA (sgRNA) that enables the controlled stoichiometry of CRIS

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

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

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

212 re, we modified the CRISPR single-guide RNA (sgRNA) to carry two distinct molecular beacons (MBs) tha

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

214 Cas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from ins

215 to constitutively express single-guide RNA (sgRNA) transcripts.

216 pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target.

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

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

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

220 is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage.

221 -124C was achieved using a single guide RNA (sgRNA)-guided and catalytically impaired Campylobacter j

222 e no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in gen

223 le consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active sta

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

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

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

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

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

229 anging the sequence of the single guide RNA (sgRNA).

230 yoblast cell lines using a single-guide RNA (sgRNA).

231 Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated

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

233 es by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors

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

235 recognizes two different single guide RNAs (sgRNA).

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

237 ded with both Cas9 vector and one of several sgRNA vectors programmed to target different test genes

238 st cell growth and to construct an in silico sgRNA library spanning the human genome.

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

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

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

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

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

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

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

246 system acts directly on each target-specific sgRNA, it enables new applications that require differen

247 It also accommodates staggered sgRNA sequences.

248 the protein regions associated with a strong sgRNA dropout effect in the screens.

249 Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan

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

251 Comparisons with other studies suggest that sgRNA auto-processing may be a phenomenon not unique to

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

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

254 can be selectively increased by changing the sgRNA target location.

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

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

257 single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex.

258 corporate cell-specific information into the sgRNA design, we develop novel interpretable machine lea

259 n through insertion of RNA aptamers into the sgRNA.

260 corporating MS2 or PP7 RNA aptamers into the sgRNA.

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

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

263 ated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well

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

265 l and tightly constrained to one side of the sgRNA binding site.

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

267 Refer to Steps 37-39 for NGS analysis of the sgRNA distribution." This step should refer the reader t

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

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

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

271 vely narrow, 300 bp window downstream of the sgRNA targets.

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

273 issues with the first 30 nucleotides of the sgRNA, which run in the opposite direction, corrections

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

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

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

277 hygromycin-resistance marker present on the sgRNA vector.

278 transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias

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

280 synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produ

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

282 de overhangs 5', but not 3', proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at t

283 thylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Esch

284 ity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfaci

285 CRISPR-Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain

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

287 mial distribution, which is better suited to sgRNA data, CB(2) outperforms the eight most commonly us

288 dicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption.

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

290 0% within 7 d postinoculation using the TRBO-sgRNA constructs, which retained 5' nucleotide overhangs

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

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

293 Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading fr

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

295 ector integration requires just one variable sgRNA to target each gene of interest, this procedure ca

296 can cause genome-wide off-target changes via sgRNA-independent DNA deamination.

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

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

299 meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein.

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