Accepted Manuscript
Cas9-NG greatly expands the targeting scope of genome-editing toolkit byrecognizing NG and other atypical PAMs in rice
Bin Ren, Lang Liu, Shaofang Li, Yongjie Kuang, Jingwen Wang, Dawei Zhang,Xueping Zhou, Honghui Lin, Huanbin Zhou
PII: S1674-2052(19)30123-6DOI: https://doi.org/10.1016/j.molp.2019.03.010Reference: MOLP 761
To appear in: MOLECULAR PLANTAccepted Date: 23 March 2019
Please cite this article as: Ren B., Liu L., Li S., Kuang Y., Wang J., Zhang D., Zhou X., Lin H., andZhou H. (2019). Cas9-NG greatly expands the targeting scope of genome-editing toolkit by recognizingNG and other atypical PAMs in rice. Mol. Plant. doi: https://doi.org/10.1016/j.molp.2019.03.010.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.
All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they arepublished as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts oruncorrected proofs.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
1
Cas9-NG greatly expands the targeting scope of genome-editing toolkit by 1
recognizing NG and other atypical PAMs in rice 2
3
Bin Ren1, 2, †, Lang Liu2, †, Shaofang Li3, Yongjie Kuang2, Jingwen Wang2, Dawei 4
Zhang1, Xueping Zhou2, 4, Honghui Lin1, * & Huanbin Zhou2, * 5
6
1Ministry of Education Key Laboratory of Bio-Resource and Eco-Environment, 7
College of Life Sciences, Sichuan University, Chengdu 610065, China. 8
2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of 9
Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China. 10
3State Key Laboratory of Plant Physiology and Biochemistry, College of Biological 11
Sciences, China Agricultural University, Beijing 100193, China 12
4State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang 13
University, Hangzhou 310058, China. 14
15
†These authors contributed equally to this work. 16
*Corresponding authors: Huanbin Zhou; E-mail: [email protected]; Phone: 17
86-10-62815914; Fax: 86-10-62894642 or Honghui Lin; Email: [email protected]; 18
Phone: 86-28-85411792; Fax: 86-28-85415300 19
20
Running title: Targeted genome engineering in rice with Cas9-NG 21
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
2
Abstract 22
The CRISPR technologies enabling precise genome manipulation are valuable for 23
the gene function study and molecular crop breeding. However, the requirement of a 24
protospacer adjacent motif (PAM), such as NGG, TTN, etc., for Cas protein 25
recognition restricts the targetable genomic loci in applications. Very lately, 26
Cas9-NG which recognizes a minimal NG PAM was reported to expand the targeting 27
space in genome editing in human cells, but little is known about its applications in 28
plants. Here, we evaluated the nuclease activity of Cas9-NG toward various NGN 29
PAMs by targeting endogenous genes in transgenic rice, and revealed that Cas9-NG 30
edits all NGG, NGA, NGT and NGC sites with impaired activities, and gene-edited 31
plants were dominated by mono-allelic mutations. The Cas9-NG-engineered base 32
editors were further developed and used to generate OsBZR1 gain-of-function plants, 33
which other available Cas9-engineered base editors are not able to. Moreover, 34
Cas9-NG-based transcriptional activator efficiently up-regulated the expression of 35
endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes 36
NAC, NTG, NTT, NCG apart from NG PAM. All these findings show that Cas9-NG 37
greatly expands the targeting scope of genome-editing tools and imply great 38
potentials for targeted genome editing, base editing and genome regulation in plants. 39
40
Key words: CRISPR, Cas9-NG, PAM, gene editing, base editing, Oryza sativa L. 41
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
3
Introduction 42
The gene editing tools, especially the Clustered Regularly Interspaced Short 43
Palindromic Repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) system 44
derived from the microbial adaptable immune systems in Streptococcus pyogenes 45
against foreign DNA sources such as bacteriophages or plasmids, have been 46
successfully applied in targeted genome editing in a wide range of plant species 47
including rice, wheat, maize, soybean, tomato, Arabidopsis, etc., showing a powerful 48
potential to advance plant biological research and crop breeding (Cermak et al., 2015; 49
Hille et al., 2018; Wang et al., 2016; Wright et al., 2016; Yin et al., 2017; Zhou et al., 50
2014). The revolutionized CRISPR/Cas9 system, as we know it today, is composed 51
of an endonuclease protein Cas9 and a single-guide RNA (sgRNA) which can 52
program Cas9 to cleave genomic DNA at a specific site and lead to a 53
double-stranded DNA break (DSB) (Hille et al., 2018). The specificity of the 54
Cas9/sgRNA-induced DNA cleavage is determined by both the PAM recognition of 55
SpCas9, a NGG sequence immediately downstream of the target region, and the 56
complementary base pairing between the sgRNA with the target DNA sequence 57
(Jiang et al., 2013). As a result, DSBs stimulate the DNA repair mechanisms in plant 58
cells and can be repaired either by error-prone nonhomologous end joining (NHEJ), 59
introducing small insertions or deletions (Indels) at the break site, or by high-fidelity 60
homology directed repair (HDR) when a homologous repair template is available, 61
introducing desired sequence changes to the target locus (Schmidt et al., 2015; 62
Waterworth et al., 2011). In recent years, many efforts have been directed toward the 63
development of Cas9 endonuclease-based systems which are suitable for gene 64
knockout, knock-in or DNA fragment deletion in plants (Lu et al., 2017; Meng et al., 65
2017; Scheben et al., 2017; Zhou et al., 2014). 66
In addition to genomic DNA manipulation which utilizes DSBs created by Cas9 67
endonuclease, programmable editing of a specific target base in plant genomic DNA 68
without requiring DSBs, termed targeted base editing, has been developed as well by 69
fusing Cas9 nickase (Cas9n) to nucleoside deaminases. In this regard, a cytidine 70
deaminase such as rat APOBEC1, human AID, lamprey CDA1, etc. or an adenine 71
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
4
deaminase evolved from E.coli TadA is directed by the Cas9n/sgRNA complex to the 72
target locus, it turns cytosine (C) or adenine (A), within the editing window in the 73
single-stranded DNA bubble created by Cas9n, into Uracil (U) or inosine (I) through 74
hydrolysis reaction, resulting in thymine (T) or guanine (G), after DNA replication 75
or repair. (Hua et al., 2018; Li et al., 2017a; Ren et al., 2018; Shimatani et al., 2017; 76
Yan et al., 2018). In most cases, a uracil glycosylase inhibitor (Nishizawa-Yokoi et 77
al.) is also tethered to Cas9n in cytosine base editors (CBEs) in order to increase the 78
base editing efficiency and outcome fidelity (Komor et al., 2016; Ren et al., 2018). 79
Both the CBEs and adenine base editors (ABEs), which enable four types of 80
nucleotide conversions (C to T, T to C, A to G, and G to A) nowadays and have been 81
continually updated, hold great promise for generating gain-of-function mutants or 82
novel germplasms in molecular crop breeding in future. 83
Autonomous transcription activation domains such as herpes simplex 84
virus-derived VP64, Xanthomonas TALE, plant-specific EDLL, ERF2m, etc. can be 85
also tethered to the catalytically inactive Cas9 protein (dCas9) to generate synthetic 86
transcriptional activators, which can be employed to reprogram the transcriptional 87
regulation of endogenous genes in nature context in plants by targeting specific DNA 88
sequences in the promoter region (Li et al., 2017b; Piatek et al., 2015; Vazquez-Vilar 89
et al., 2016). Robust transcriptional activation of single or multiple target genes 90
could be achieved in planta depending on the positional targeting of dCas9 to the 91
promoter and/or the synergistic or additive effects of multiple sgRNAs targeting the 92
same region (Lowder et al., 2015; Piatek et al., 2015). dCas9-based transcription 93
regulators further expand the CRISPR toolkit for gene function study in plants. 94
Although the CRISPR/Cas9-related technologies have been widely adopted for 95
genome editing in plants due to its high efficiency, specificity, simplicity and 96
versatility, the need of a specific PAM, usually NGG and sometimes NAG, for Cas9 97
recognition restricts the targeting range of these tools, especially base editors, given 98
that it requires a functional PAM for Cas9n interaction to localize the target base in 99
the editing window within the protospacer for deamination (Li et al., 2018a; Mao et 100
al., 2013; Meng et al., 2018; Zong et al., 2017). To address this problem, several 101
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
5
approaches were adopted to expand, to a certain extent, the targeting scope of these 102
CRISPR tools by using a number of engineered Cas9 variants, naturally occurring 103
orthologues and other Cas proteins with altered PAM specificities, such as VQR, 104
EQR, VRER SpCas9 variants for NGA, NGAG, NGCG PAMs (Kleinstiver et al., 105
2015; Ren et al., 2017), Staphylococcus aureus Cas9 (SaCas9) for NNGRRT (Hua et 106
al., 2019; Ran et al., 2015), Neisseria meningitides Cas9 (NmCas9) for NNNNGATT 107
(Esvelt et al., 2013), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) for TTTV (Li et 108
al., 2018b; Zetsche et al., 2015), etc. it is, however, still one of the major limitations 109
waiting to be addressed for applications of CRISPR technologies in plants to date. 110
Recently, a number of engineered Cas9 variants with the broadest PAM 111
compatibility in our knowledge were reported. Among them, Cas9-NG, xCas9 and 112
its relevant fusion proteins showed remarkable advancements in gene editing and 113
transcriptional regulation without sacrificing the DNA specificity in human cells (Hu 114
et al., 2018; Nishimasu et al., 2018), but whether and how it improves DNA 115
recognition capabilities in plants have not been investigated in detailed yet. In this 116
study, the efficacy of both Cas9-NG and xCas9 endonucleases toward the NGN PAM 117
targets was evaluated in transgenic rice plants. We demonstrate that Cas9-NG, 118
outperforming xCas9, efficiently induces mutations of target genes at NGG, NGA 119
and NGT PAM sites in rice, and it greatly expands the scope of base editors and 120
transcriptional activator with an efficiency. Notably, Cas9-NG recognizes a diverse 121
set of atypical PAM sequences in addition to NG PAM. All the data imply great 122
potentials in applications of Cas9-NG and its related tools in rice genome 123
manipulation. 124
125
Results 126
Cas9-NG induces Indel mutations at NGN PAM sites in rice protoplasts 127
To explore the potentials of those engineered Cas9 variants, which has been reported 128
with broad PAM compatibility in human cells, in rice genome editing applications, 129
we first assessed its cleavage activity toward endogenous genomic loci in rice cells. 130
The Cas9 variant carrying 131
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
6
R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R mutations, namely 132
Cas9-NG, was codon-optimized for expression in rice and attached with nuclear 133
localization signals (NLSs) at both termini (Supplemental Table 1 and Figure 1A). 134
The Cas9-NG was driven by the CaMV 35S promoter and the sgRNAs were under 135
control of the rice U6 gene promoters as mentioned previously (Jiang et al., 2013). 136
Both Cas9-NG and individual sgRNA targeting a BamHI restriction site in OsGSK4, 137
OsCERK1 or OsETR2 toward an NGG, NGA, NGT or NGC PAM were 138
co-expressed in rice leaf sheath protoplasts (Figure 1B-1D; Supplemental Figure 1; 139
Supplemental Table 2). Genomic DNA was extracted from pooled cells at 48 h 140
post-transfection, and treated with BamHI to enrich the mutated alleles which carried 141
the destroyed BamHI-recognition sites resulting from Cas9-NG cleavages. The 142
BamHI-resistant genomic DNA was used for PCR amplification with gene-specific 143
primers. Amplicons were further cloned, and positive colonies which were identified 144
by BamHI digestion were chosen randomly for sequencing. 145
As for the OsGSK4 target site which carries a canonical NGG PAM for the 146
wild-type Cas9, sequencing of 15 clones revealed 8 distinct mutations, with 147
deletions and/or insertions located in close proximity to the predicted cleavage site 148
of the Cas9-NG/sgRNA complexes (Supplemental Figure 1). It quite resembles the 149
outcome of Cas9 nuclease in the PCR/restriction enzyme assays reported previously 150
(Jiang et al., 2013). For the NGA and NGT target sites, the frequencies of mutations 151
induced by Cas9-NG in OsCERK1 and OsGSK4 were comparable to and even higher 152
than that for NGG (Figure 1B and 1C). Notably, Cas9-NG showed low editing 153
efficiency at the NGC target site given that BamHI-undigested target region was not 154
easy to be enriched and only 2 mutated versions were discovered in our experiment 155
(Figure 1D). Collectively, these data show that Cas9-NG endonuclease can cleaves 156
NGG, NGA and NGT targets, whereas it has poor activity toward NGC target in rice. 157
Targeted gene knockout with Cas9-NG nuclease in transgenic rice plants 158
To validate the capacity and efficacy of Cas9-NG endonuclease in targeted gene 159
knockout in rice, we further tested Cas9-NG in stable transgenic rice plants. In this 160
case, the Cas9 gene in the binary vector pUbi:Cas9 (Zhou et al., 2014) was replaced 161
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
7
by the codon-optimized Cas9-NG, resulting in pUbi:Cas9-NG. Alternatively, another 162
evolved Cas9 variant, which carries 163
A262T/R324L/S409I/E480K/E543D/M694I/E1219V mutations and also recognizes 164
NG PAM in human cells (Hu et al., 2018), was chosen to be codon-optimized and 165
constructed, resulting in pUbi:xCas9 (Supplemental Table 1). Custom sgRNAs can 166
be easily shuttled into those binary vectors through Gateway recombination. 167
Cas9-NG, xCas9 and Cas9 were tested side-by-side with the same sgRNAs which 168
target OsMPK11 with a NGG PAM, OsMPK7 with a NGA PAM, OsMPK10 with a 169
NGT PAM or OsMPK8 with a NGC PAM (Supplemental Table 3). After 170
transformation of the Cas/sgRNA constructs in rice callus and regeneration of 171
transgenic plants, mutations of each target gene have been detected by directly 172
sequencing the PCR amplicons of the target regions (Supplemental Table 4). 173
In T0 generation, approximately 38.24% (13 out of 34 lines) of independent 174
transgenic rice lines expressing Cas9-NG were identified with Indels at the canonical 175
NGG target site in OsMPK11. Of which, 11 lines contained mono-allelic mutations 176
(Figure 2A). By contrast, the frequency of Indels caused by Cas9 together with the 177
identical sgRNA was approximately 82.14% (23 out of 28 lines) and 21 lines were 178
identified as di-allelic (Supplemental Figure 2A). However, xCas9 showed 179
dramatically reduced Indel frequency for this NGG target site (Supplemental Figure 180
2B). At the NGA PAM site in OsMPK7, Cas9-NG resulted in 8.3-fold increase of 181
mutation frequency, 25% Indels compared to around 3% for Cas9 and xCas9, in 182
which mono-allelic mutations dominated (Figure 2B; Supplemental Figure 2C and 183
2D). Meanwhile, Cas9-NG achieved approximately 21.43% (9 out of 42 lines) Indels 184
at the NGT PAM sites in OsMPK10 and 4.55% (2 out of 44 lines) Indels at the NGC 185
PAM sites in OsMPK8, respectively, while Cas9 and xCas9 yielded no detectable 186
mutations at both target sites (Figure 2C and 2D). Next, we investigated the 187
off-target effects of Cas9-NG. Three and two potential off-target sites corresponding 188
to OsMPK10- and OsMPK11-targeting sgRNA, respectively, were chosen to be 189
examined in transgenic plants in terms of the high level of homology in 190
bioinformatics analysis. Interestingly, cleavage activity of Cas9-NG was detected 191
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
8
only in the off-target gene OsMPK9. In this regard, a single mismatch locates in the 192
PAM sequence, resulting in NGC PAM instead of NGT PAM for Cas9-NG 193
recognition. As a result, approximately 9.52% (4 out of 42 lines) Indels in OsMPK9, 194
independent of or together with on-target mutations in individual plant, was achieved 195
by the OsMPK10-targeting sgRNA (Supplemental Figure 3A-3C). Combined all data 196
together (Figure 2E), we conclude that Cas9-NG efficiently edits NGG, NGA and 197
NGT targets, albeit with low activity on NGC, greatly expanding the scope of 198
genome editing in rice comparing to xCas9. 199
Moreover, we have investigated whether Cas9-NG could be used in multiplex 200
genome editing in rice plants. Two sgRNAs targeting OsSERK1 and OsSERK2 201
simultaneously toward NGG PAM were transferred into rice callus together with 202
Cas9, Cas9-NG or xCas9 using Agrobacterium-mediated transformation, and 203
regenerated plants were genotyped by sequencing as mentioned above. We found 204
that 10 lines expressing Cas9 were identified with Indels, accounting for 83.33% of 205
all T0 lines. All plants were characterized as double gene mutants and were di-allelic 206
at both NGG target sites (Figure 3C; Supplemental Figure 4A). By contrast, 207
Cas9-NG showed slightly higher editing efficiency whereas mono-allelic mutations 208
occurred dominantly at both sites (Figure 3A and 3C). xCas9 was less active given 209
that 16.28% of OsSERK1 and 60.47% of OsSERK2 were mutated in T0 lines, 210
respectively (Supplemental Figure 4B). Meanwhile, two sgRNAs targeting OsGSK4 211
and OsETR2 simultaneously toward non-canonical NGT PAM were tested for 212
Cas9-NG as well. 45 out of 47 independent lines (95.83% frequency) were identified 213
with Indels. Among them, 6 were double mutants whereas the remaining was single 214
mutant of OsGSK4 (Figure 3B and 3C). Collectively, these data suggest Cas9-NG 215
can be used for efficient multiplex gene editing toward NG PAM in rice, but the 216
mutation efficiency is highly variable at different loci. 217
Cytosine and adenine base editing with Cas9-NG in rice 218
In addition to inducing target gene mutation, CRISPR/Cas systems can be adapted 219
for precise base editing in various plant species as well. Therefore, we engineered 220
Cas9-NG nuclease into Cas9-NG D10A nickase and used it to replace the Cas9n 221
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
9
gene in the well-established CBE architecture (rBE9) (Ren et al., 2018), resulting in 222
rBE22 (Figure 4A). The base editing activity of rBE22 was first tested in rice 223
protoplasts with the sgRNA targeting an ApaLI restriction site in OsRLCK185 224
toward NGC PAM using the PCR/RE method mentioned above, and C>T 225
conversions in the editing window were detected as anticipated (Figure 4B), 226
suggesting Cas9-NG is compatible with the base editor architecture. 227
BZR1 is the master transcription factor of the BR signaling pathway and the 228
key integration node of numerous signaling cascades in Arabidopsis, whose 229
gain-of-function allele carrying P234L mutation has been engineered for the 230
resistance to thrip feeding in Lotus japonicas and fruit quality improvement in 231
tomato (Liu et al., 2014; Miyaji et al., 2014). Therefore, engineering its homologue, 232
OsBZR1 in rice, in the same way could offer plant breeders a novel germplasm for 233
applications in agriculture. To achieve this goal, we first tried rBE5 toward a NAG 234
PAM in the target region but failed (Supplemental Figure 5A). Further, we designed 235
a sgRNA in relation to a NGA PAM (Supplemental Figure 5B) and delivered it with 236
rBE22 into rice callus. Four lines were identified but with unwanted mutations from 237
45 regenerated plants being sequenced (Supplemental Figure 5B and 5C). To be 238
noted, with the same sgRNA, no activities at all were detected for rBE9 and rBE20, 239
which were constructed with Cas9 and xCas9, respectively. At the end, the third 240
sgRNA in regarding to a NGT PAM (Figure 4C) was utilized with rBE22 to edit 241
OsBZR1. Surprisingly, approximately 35.29% frequency of base editing, including 242
the nucleotide change of interest, was obtained by screening of 51 transgenic plants 243
(Figure 4D; Supplemental Figure 5D and 5E). 244
A pathogen-responsive phosphorylation site in OsSERK2 was also tested for 245
rBE22’s activity toward the NGT PAM in transgenic rice plants. Only one line was 246
identified with base editing event (Supplemental Figure 6A-6C). 247
Meanwhile, the adenine base editor rBE14 (Yan et al., 2018) was updated with 248
Cas9-NG as well, the resulting rBE23 enabled A>G conversion in rice protoplast as 249
expected (Figure 4E and 4F). A previously characterized target site in OsSERK2 was 250
tested in regard to the NGA PAM, approximately 40.43% (19 out of 47 lines) of 251
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
10
independent transgenic rice lines expressing rBE23 were identified with nucleotide 252
changes at the NGA PAM site in OsSERK2 (Figure 4G-4I; Supplemental Figure 6D), 253
which is slightly higher than that for rBE14 in the case of NGG PAM reported 254
previously (Yan et al., 2018). In addition, the same sgRNA was further used to test 255
rBE22’s activity, 54.16% frequency of base editing in a wide editing window of the 256
target region was detected (Figure 4J; Supplemental Figure 6D). Combined all the 257
data together (Figure 4K), we conclude that Cas9-NG nickase, which recognizes the 258
minimal NG PAM, greatly expands the number of target sites for base editing in rice 259
in contrast to other reported CRISPR/Cas systems. 260
Transcriptional regulation of endogenous genes by engineered Cas9-NG 261
activation complexes in rice 262
Many efforts have been directed toward targeted genome regulation in recent years. 263
It’s a powerful approach in gene function study. To investigate whether Cas9-NG is 264
suitable for using in programmable transcriptional activation, we generated the 265
nuclease-inactive Cas9-NG (D10A/H840A, dCas9-NG) and then engineered with 266
TAD, which was modified from the TV domain (Li et al., 2017b) with XTEN linkers 267
as spacers between each TAL repeat, resulting in dCas9-NG-TAD (Figure 5A; 268
Supplemental Table 1). At the same time, dCas9-TAD was also constructed using the 269
same strategy. 270
The synthetic activators were first tested with sgRNAs targeting the antisense 271
strand of the endogenous OsCOI2 gene promoter region toward either NGG (-181 bp 272
upstream from the transcription start site/TSS) or NGA PAM (-123 bp from the TSS), 273
relative transcript abundances of OsCOI2 in transgenic rice plants were measured 274
using qRT-PCR (Figure 5B). Regarding the NGG PAM, dCas9-NG-TAD had slightly 275
less ability of transcriptional activation in contrast to dCas9-TAD, given that the 276
expression of OsCOI2 was obviously induced 3-6-fold by both activators in certain 277
individuals (Figure 5C), but it was highly variable in all dependent lines generated 278
by dCas9-NG-TAD (Data not shown). However, high-level transcriptional activation 279
of OsCOI2 in regard to the NGA PAM was much more readily detected in transgenic 280
plants expressing dCas9-NG-TAD, compared to that for dCas9-TAD (Figure 5C). 281
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
11
Furthermore, a sgRNA related to NGT PAM was designed to target the sense strand 282
of the OsSERK2 promoter region at sites of -176 bp upstream from the TTS (Figure 283
5D), the results showed that dCas9-NG-TAD, but not dCas9-TAD, activated 284
OsSERK2 expression by high-fold. Meanwhile, another NGG target site was chosen 285
to induce the expression of OsSERK2, and the transcriptional induction was quite 286
similar to that of OsCOI2 (Figure 5E). Collectively, these data suggest that the 287
nuclease-inactive Cas9-NG as well as dCas9, endowed with gene activation ability, 288
can be universally employed for activation of endogenous genes in rice. 289
Cas9-NG recognizes atypical PAM sequences other than NG in rice 290
Cas9-NG has been engineered from the wild-type Cas9 with substitutions of seven 291
amino acid residues related to PAM interaction, it recognizes more flexible PAM 292
sequences besides NG, while slightly sacrificing the enzymatic activity (Nishimasu 293
et al., 2018). However, the requirement of atypical PAMs has not been investigated 294
in eukaryote cells yet. To further explore the DNA specificity of Cas9-NG, we tested 295
the cleavage activity of Cas9-NG endonuclease toward all 16 possible alternate PAM 296
sequences, varying at the second and third positions, by targeting 16 genomic loci in 297
rice protoplasts using the PCR/RE method (Supplemental Table 2 and 4). As a result, 298
4 distinct PAM sequences, including NAC, NTG, NTT and NCG, were repeatedly 299
identified, given that Indels occurred at the corresponding restriction sites 300
(Supplemental Figure 7A-7E). To confirm the facticity of these novel PAMs for 301
Cas9-NG recognition, we delivered Cas9-NG into rice calli, together with sgRNAs 302
targeting OsGSK4, Os03g02040 or OsCERK1 related to each identified PAMs, 303
respectively. After genotyping the regenerated plants, approximately 8.51%, 8.33%, 304
4.17% and 11.63% frequencies of Indels were detected at the NAC, NTG, NTT and 305
NCG PAM target sites, respectively (Figure 6A-6E). Again, mono-allelic mutations 306
dominated in these plants. Therefore, we presume that Cas9-NG recognizes more 307
PAMs in rice than previously thought, at least NG, NAC, NTG, NTT and NCG in 308
our case. The broad PAM compatibility of Cas9-NG enables its related genome 309
editing tools to be more widely utilized. 310
311
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
12
Discussion 312
In this study, activities of Cas9-NG, xCas9 and Cas9 at a number of endogenous loci 313
bearing different PAMs have been fully investigated for targeted gene knockout, 314
base editing as well as transcriptional activation in transgenic rice plants, 315
respectively. We show that Cas9-NG recognizes a broad range of PAM sequences, 316
including NG, NAC, NTG, NTT, NCG, with moderately impaired nuclease activity. 317
Accordingly, its relevant tools enable modification of previously insensitive sites in 318
rice genome. All the results imply that Cas9-NG greatly expands the targeting scope 319
of genome editing tools. 320
During CRIPSR/Cas-mediated DNA cleavage, Cas protein initially interacts 321
with PAM sequence, which triggers local DNA melting, sgRNA invasion and DSBs 322
at the target sites eventually (Jiang and Doudna, 2017). The presence of PAM is 323
critical for the accessibility of Cas proteins to the target region for genome 324
engineering. In other words, it restricts the targeting range of CRISPR/Cas tools 325
especially for applications that require precise positioning of Cas proteins. To date, 326
many Cas proteins and its variants with different PAM specificities have been 327
identified and characterized experimentally in a broad range of organisms and cell 328
types, like SpCas9 (NGG), SpCas9VQR (NGA), SaCas9 (NNGRRT), LbCas12 329
(TTN) etc (Cong et al., 2013; Kleinstiver et al., 2015; Zetsche et al., 2015). Among 330
them, SpCas9 is widely adapted in various genome editing tools since none of other 331
Cas proteins offer a PAM that occurs as frequently as that of SpCas9. In an effort to 332
overcome the PAM limitation of SpCas9, xCas9, the TLIKDIV variants of SpCas9, 333
has been evolved using the phage-assisted continuous evolution method, resulting in 334
recognize NG, GAA and GAT PAM, and it has been proved to expand the DNA 335
targeting scope of CRISPR systems in mammalian cells (Hu et al., 2018). However, 336
consistent with the recent report that the efficiency of xCas9 is strikingly lower than 337
that of Cas9 at NG PAM sites in rice (Wang et al., 2018), we found that xCas9 had 338
extremely weak and no nuclease activities at the NGA, NGT and NGC site under our 339
assay conditions, respectively, albeit with low and variable efficiency at the original 340
NGG sites tested. Notably, most of xCas9-editing plants carried mono-allelic 341
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
13
mutation, in contrast to di-allelic mutations for Cas9. Consequently, xCas9-based 342
CBE has failed in base editing at a number of target sites in transgenic rice plants 343
(Data not shown). 344
In comparison, Cas9-NG exhibited great abilities in the improvement of 345
genome editing tools in our study. Cas9-NG is the VRVRFRR variant of SpCas9, in 346
which Arg1335 is mutated to Valine to eliminate the interaction between Arg1335 347
and the third G in the PAM during PAM recognition, and other amino acid 348
substitutions facilitate the PAM interactions and partially compensate the lost 349
enzymatic activity caused by Arg1335 mutation, resulting in recognizing NG PAM 350
(Nishimasu et al., 2018). In our study, Cas9-NG achieved 38.24-93.33% editing 351
efficiency at the tested NGG PAM sites compared to over 82% for Cas9. The 352
nuclease activity of Cas9-NG was presumed to be impaired because high percentage 353
of mono-allelic mutation occurs in the mutant plants. Even though, Cas9-NG 354
achieved 25% editing efficiency at the NGA PAM site compared to 3.33% for Cas9, 355
12.5-95.83% editing efficiency at the NGT PAM sites as well as 4.55% editing 356
efficiency at the NGC PAM site which Cas9 is not accessible, respectively. 357
Therefore, we presume that Cas9-NG can efficiently edit NGG, NGA and NGT sites 358
in rice genome, while it has relatively low activity toward NGC. More NGN target 359
sites, especially NGC site, should be investigated in the future. More interestingly, 360
Cas9-NG recognizes at least 4 types of atypical PAMs (NAC, NTG, NTT and NCG) 361
with efficiency range from 4.17-11.63% in rice plants, which is comparable to or 362
even higher than that for NGC PAM. These PAMs have not been tested in human 363
cells yet, but both NAC and NTG PAM sequences were detected in off-target 364
mutations when editing EMX1 by Cas9-NG (Nishimasu et al., 2018). Therefore, the 365
PAM specificity of Cas9-NG is more complicated than previously thought, detailed 366
PAM requirements of Cas9-NG should be carried out in eukaryotic cells in the future. 367
Moreover, guide RNAs should be designed carefully to avoid off-target effects in use 368
of Cas9-NG. 369
Generally speaking, PAM compatibility of Cas protein is the key point of base 370
editor since it requires precise Cas positioning to locate the target nucleotide within 371
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
14
the editing window in base editing. In our study, OsBZR1 gain-of-function mutants 372
carrying C>T conversions have been successfully identified with high efficiency by 373
chromosome walking thanks to the broad PAM flexibility of Cas9-NG. Meanwhile, 374
the Cas9-NG-based ABE yielded A>G conversions in OsSERK2 at 40.43% 375
efficiency, slightly higher than that for Cas9-based ABE. Therefore, we include that 376
the amino acid substitutions of Cas9-NG do not affect the editing efficiency of base 377
editors. On the other hand, it reduces the nickase activity, resulting in higher fidelity 378
of base editing outcomes. Furthermore, a genome-wide prediction of targetable 379
nucleotides indicated the coverages of rBE22 and rBE23 were 8-fold more than that 380
of rBE9 and rBE14 in rice (Data not shown). Moreover, Cas9-NG is compatible with 381
TAD as well, capable of up-regulating the expressions of OsCOI2 and OsSERK2 to 382
the same level as Cas9 did. Thus, all the data imply that Cas9-NG, with more target 383
sites accessible, can serve as a useful RNA-guided DNA targeting platform for 384
numerous effectors, like transcriptional repressors, DNA recombinases, epigenetic 385
enzymes etc. in rice. 386
Very lately, another group reported that the preliminary version Cas9-NGv1 387
with ARVRFRR mutations could mutagenize endogenous target sites with NGN 388
PAM in both the rice and Arabidopsis genomes (Endo et al., 2019), but the true 389
efficiency of Cas9-NGv1 for each target site are not available given that they 390
calculated the percentages of positive clones in regard to specific calli. To be noted, 391
the cleavage kinetics of Cas9-NGv1 is slower than that of Cas9-NG, this is most 392
likely due to Val1335 in Cas9-NG instead of Ala1335 in Cas9-NGv1, which 393
stabilizes the interaction between Arg1333 and the second G in the PAM (Nishimasu 394
et al., 2018). Here, the advanced version Cas9-NG has been focused on, a set of 395
atypical PAMs other than the reported NG PAM has been identified, giving novel 396
insights into the study of Cas9-NG. We believe that the detailed investigation of 397
Cas9-NG in our study will facilitate the applications of the Cas9-NG toolkit in rice 398
and the development of similar tools for genome editing in other plant species. 399
400
Methods 401
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
15
Vector design and plasmid construction 402
The coding regions of xCas9 (Hu et al., 2018) and Cas9-NG (Nishimasu et al., 2018) 403
flanked with two nuclear localization signal (NLS) sequences were codon-optimized 404
for improved expression in rice and individually synthesized by Tsingke 405
(Supplemental Table 1). It was directly cloned downstream of the maize ubiquitin 1 406
promoter and upstream of the NOS terminator by replacing the Cas9 gene in 407
pUC19:Cas9 and pUbi:Cas9 (Zhou et al., 2014) with BamHI/SpeI digestion and 408
DNA ligation, respectively, resulting in pUC19:xCas9 and pUC19:Cas9-NG for 409
transient expression analysis in rice protoplasts, as well as pUbi:xCas9 and 410
pUbi:Cas9-NG for stable rice transformation. 411
To construct base editors, gene fusions of each Cas9 variant and deaminase 412
encoding genes were first carried out with a simple PCR-based cloning strategy, and 413
all the primers used are listed in Supplemental Table 4. Briefly, the rBE9 gene 414
fragment (Ren et al., 2018) was subcloned into pUC19, by EcoRI/SpeI digestion and 415
DNA ligation, resulting in pUC19:rBE9. The partial fragments of xCas9 and 416
Cas9-NG were PCR amplified with the high-fidelity DNA polymerase Phusion 417
(NEB) and the primer pairs Cas9-Fg1-F1/Cas9-Fg2-R1, and ligated with the 418
backbone of pUC19:rBE9 which was amplified with the primer pairs 419
UGI-F1/hAID-R1, resulting in pUC19:rBE20 and pUC19:rBE22, respectively. In the 420
same manner, the partial Cas9-NG fragments were amplified with the primer pairs 421
Cas9-Fg1-F1/NLS-R2 and ligated with the backbone of pUC57:TadA-TadA7.10 422
(Yan et al., 2018) which was amplified with the primer pairs pUC57-F1/TadA-R1, 423
resulting in pUC57:rBE23. The identities of all chimeric genes in the correct 424
orientation were confirmed by sequencing. Finally, rBE20, rBE22 and rBE23 were 425
used to replace the Cas9 fragment in the vector pUbi:Cas9 (Zhou et al., 2014) as 426
mentioned above, to create the destination binary vectors pUbi:rBE20, pUbi:rBE22 427
and pUbi:rBE23 for targeted base editing in rice, respectively. 428
To construct vectors for transcriptional activation of endogenous genes in rice, 429
the Cas9 and Cas9-NG were first mutated into dCas9 and dCas9-NG, the 430
catalytically inactive version carrying D10A and H840A mutations, by PCR-based 431
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
16
mutagenesis using pUC57:Cas9 and pUC57:Cas9-NG as the templates, respectively. 432
The TAD fragment, modified from dCas9-TV (Li et al., 2017b) and encoding a 433
fusion protein domain which is consist of 6 TALE TAD motifs interspersed with 434
XTEN linkers and 8 tandem repeats of VP16, was codon-optimized and synthesized 435
by Tsingke (Supplemental Table 1). The dCas9, dCas9-NG and TAD gene fragments 436
were amplified from the aforementioned plasmids by PCR using the primer pairs 437
Cas9-F1/Cas9-Fg2-R1 and TAD-F1/pUC57-R1, respectively, and assembled 438
together to create pUC57:dCas9-TAD and pUC57:dCas9-NG-TAD. Chimeric gene 439
identity was confirmed by sequencing. Finally, dCas9-TAD and dCas9-NG-TAD 440
fragments were individually cloned into the binary vectors by Cas9 replacement as 441
mentioned above, resulting in pUbi:dCas9-TAD and pUbi:dCas9-NG-TAD. All the 442
primers used are listed in Supplemental Table 4. 443
The final T-DNA transformation plasmids were constructed as previously 444
described (Yan et al., 2018). Briefly, for the target site of each gene (Supplemental 445
Table 2 and 3), the complementary oligos (Supplemental Table 4) with appropriate 446
4-bp overhangs were phosphorylated (PNK, NEB), annealed and then cloned into 447
the entry vector pENTR:sgRNA8 (Yan et al., 2018) at the BsaI or BtgZI cutting site. 448
The sgRNA expression cassettes were finally shuttled into appropriate destination 449
vectors by LR clonase (Invitrogen). 450
Rice transformation 451
Rice cultivar Kitaake (Oryza sativa spp. Geng) was used in this study. The 452
Cas9/sgRNA-, xCas9/sgRNA-, Cas9-NG/sgRNA-, rBE/sgRNA- and 453
Cas9-NG-TAD/sgRNA-expressing binary plasmids were introduced into 454
Agrobacterium tumefaciens strain EHA105 by electroporation, and then used for rice 455
transformation with immature seed-derived calli as described previously (Hiei et al., 456
1994). 457
Genomic DNA cleavage detection in rice protoplasts 458
Isolation and transfection of rice leaf sheath protoplasts were conducted as 459
previously reported (Jiang et al., 2013). Briefly, a total of 10 µg of Cas9, xCas9 or 460
Cas9-NG construct together with the corresponding sgRNA-expressing construct 461
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
17
(molar ratio 1:1) was used to transfect approximately 4 x 105 protoplasts using the 462
PEG-mediated transformation, respectively. Transfected protoplasts were incubated 463
in darkness for 48 h and then harvested for genomic DNA extraction using the 464
CTAB method (Porebski et al., 1997). Genomic DNA was predigested with BamHI 465
or XbaI according to the target sequence, and then used as the template for PCR 466
amplifying each target region of endogenous genes with I5 high-fidelity DNA 467
polymerase (MCLAB) and gene-specific primer pairs listed in Supplemental Table 4. 468
The amplicons were digested with the same restriction enzyme again, and separated 469
on an Agarose gel. Uncut bands were cloned into a pEASY-Blunt cloning vector 470
(TransGen Biotech) for DNA sequencing and Indel mutation verification. 471
On-target and Off-target mutation detection in rice plants 472
Genomic DNA was isolated from independent transgenic rice lines, and then used as 473
templates for PCR amplification with I5 high-fidelity DNA polymerase (MCLAB) 474
and gene-specific primer pairs listed in Supplemental Table 4 to amplify the genomic 475
regions containing the target sites or putative off-target sites. PCR products were 476
sequenced directly and the sequencing chromatograms were analyzed for potential 477
mutations. 478
Quantitative RT-PCR 479
Total RNA of rice leaves was extracted with TRIzolTM Reagent (Invitrogen) and used 480
to synthesize First-strand cDNAs using the PrimeScriptTM RT Reagent Kit with 481
gDNA Eraser (Takara Bio) according to the manufacturers’ instructions. qRT-PCR 482
was performed by using SYBR Premix Ex Taq II (Takara Bio) on Applied 483
Biosystems 7500 Real-Time PCR System (Applied Biosystems). The transcript 484
levels of each gene in independent transgenic lines were calculated by the relative 485
quantitation method (∆∆Ct). OsActin was used as the internal control to normalize 486
all data and sequences of the primers used in qRT-PCR are listed in Supplemental 487
Table 4. 488
489
Author Contributions 490
H.Z., H.L., X.Z., and D.Z. designed the experiments; B.R., L.L., Y.K., and J.W. 491
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
18
conducted the experiments; S.L. performed the bioinformatics analysis; H.Z. wrote 492
the paper with input of all other authors; all authors participated in discussion and 493
revision of the manuscript. 494
495
Acknowledgements 496
The authors have filed a patent application based on the results reported in this study. 497
This study was supported by grants from the National Natural Science Foundation of 498
China (31871948), the National Key Research and Development Program of China 499
(2017YFD0200900) and the Agricultural Science and Technology Innovation 500
Program of the Chinese Academy of Agricultural Sciences to H.Z. 501
502
Supplemental Information 503
Supplemental information is available in the online version of this article. 504
505
References 506
Cermak, T., Baltes, N.J., Cegan, R., Zhang, Y., and Voytas, D.F. (2015). 507
High-frequency, precise modification of the tomato genome. Genome Biol. 508
16:232. 509
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., 510
Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering 511
using CRISPR/Cas systems. Science 339:819-823. 512
Endo, M., Mikami, M., Endo, A., Kaya, H., Itoh, T., Nishimasu, H., Nureki, O., and 513
Toki, S. (2019). Genome editing in plants by engineered CRISPR-Cas9 514
recognizing NG PAM. Nat. Plants 5:14-17. 515
Esvelt, K.M., Mali, P., Braff, J.L., Moosburner, M., Yaung, S.J., and Church, G.M. 516
(2013). Orthogonal Cas9 proteins for RNA-guided gene regulation and 517
editing. Nat. Methods 10:1116-1121. 518
Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of 519
rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of 520
the boundaries of the T-DNA. The Plant J. 6:271-282. 521
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
19
Hille, F., Richter, H., Wong, S.P., Bratovič, M., Ressel, S., and Charpentier, E. (2018). 522
The biology of CRISPR-Cas: backward and forward. Cell 172:1239-1259. 523
Hu, J.H., Miller, S.M., Geurts, M.H., Tang, W., Chen, L., Sun, N., Zeina, C.M., Gao, 524
X., Rees, H.A., Lin, Z., et al. (2018). Evolved Cas9 variants with broad PAM 525
compatibility and high DNA specificity. Nature 556:57-63. 526
Hua, K., Tao, X., Yuan, F., Wang, D., and Zhu, J.K. (2018). Precise A.T to G.C base 527
editing in the rice genome. Mol. Plant 11:627-630. 528
Hua, K., Tao, X., and Zhu, J.K. (2019). Expanding the base editing scope in rice by 529
using Cas9 variants. Plant Biotechnol. J. 17:499-504. 530
Jiang, F., and Doudna, J.A. (2017). CRISPR-Cas9 structures and mechanisms. Annu. 531
Rev. Biophys. 46:505-529. 532
Jiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B., and Weeks, D.P. (2013). 533
Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene 534
modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 535
41:e188. 536
Kleinstiver, B.P., Prew, M.S., Tsai, S.Q., Topkar, V.V., Nguyen, N.T., Zheng, Z., 537
Gonzales, A.P., Li, Z., Peterson, R.T., Yeh, J.R., et al. (2015). Engineered 538
CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481-485. 539
Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., and Liu, D.R. (2016). 540
Programmable editing of a target base in genomic DNA without 541
double-stranded DNA cleavage. Nature 533:420-424. 542
Li, C., Zong, Y., Wang, Y., Jin, S., Zhang, D., Song, Q., Zhang, R., and Gao, C. 543
(2018a). Expanded base editing in rice and wheat using a Cas9-adenosine 544
deaminase fusion. Genome Biol. 19:59. 545
Li, J., Sun, Y., Du, J., Zhao, Y., and Xia, L. (2017a). Generation of targeted point 546
mutations in rice by a modified CRISPR/Cas9 system. Mol. Plant 547
10:526-529. 548
Li, X., Wang, Y., Liu, Y., Yang, B., Wang, X., Wei, J., Lu, Z., Zhang, Y., Wu, J., 549
Huang, X., et al. (2018b). Base editing with a Cpf1-cytidine deaminase 550
fusion. Nat. Biotechnol. 36:324-327. 551
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
20
Li, Z., Zhang, D., Xiong, X., Yan, B., Xie, W., Sheen, J., and Li, J.F. (2017b). A 552
potent Cas9-derived gene activator for plant and mammalian cells. Nat. 553
Plants 3:930-936. 554
Liu, L., Jia, C., Zhang, M., Chen, D., Chen, S., Guo, R., Guo, D., and Wang, Q. 555
(2014). Ectopic expression of a BZR1-1D transcription factor in 556
brassinosteroid signalling enhances carotenoid accumulation and fruit quality 557
attributes in tomato. Plant Biotechnol. J. 12:105-115. 558
Lowder, L.G., Zhang, D., Baltes, N.J., Paul, J.W., 3rd, Tang, X., Zheng, X., Voytas, 559
D.F., Hsieh, T.F., Zhang, Y., and Qi, Y. (2015). A CRISPR/Cas9 toolbox for 560
multiplexed plant genome editing and transcriptional regulation. Plant 561
Physiol. 169:971-985. 562
Lu, Y., Ye, X., Guo, R., Huang, J., Wang, W., Tang, J., Tan, L., Zhu, J.K., Chu, C., 563
and Qian, Y. (2017). Genome-wide targeted mutagenesis in rice using the 564
CRISPR/Cas9 system. Mol. Plant 10:1242-1245. 565
Mao, Y., Zhang, H., Xu, N., Zhang, B., Gou, F., and Zhu, J.K. (2013). Application of 566
the CRISPR-Cas system for efficient genome engineering in plants. Mol. 567
Plant 6:2008-2011. 568
Meng, X., Hu, X., Liu, Q., Song, X., Gao, C., Li, J., and Wang, K. (2018). Robust 569
genome editing of CRISPR-Cas9 at NAG PAMs in rice. Sci. China Life Sci. 570
61:122-125. 571
Meng, X., Yu, H., Zhang, Y., Zhuang, F., Song, X., Gao, S., Gao, C., and Li, J. 572
(2017). Construction of a genome-wide mutant library in rice using 573
CRISPR/Cas9. Mol. Plant 10:1238-1241. 574
Miyaji, T., Yamagami, A., Kume, N., Sakuta, M., Osada, H., Asami, T., Arimoto, Y., 575
and Nakano, T. (2014). Brassinosteroid-related transcription factor 576
BIL1/BZR1 increases plant resistance to insect feeding. Biosci. Biotechnol. 577
Biochem. 78:960-968. 578
Nishimasu, H., Shi, X., Ishiguro, S., Gao, L., Hirano, S., Okazaki, S., Noda, T., 579
Abudayyeh, O.O., Gootenberg, J.S., Mori, H., et al. (2018). Engineered 580
CRISPR-Cas9 nuclease with expanded targeting space. Science 581
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
21
361:1259-1262. 582
Nishizawa-Yokoi, A., Cermak, T., Hoshino, T., Sugimoto, K., Saika, H., Mori, A., 583
Osakabe, K., Hamada, M., Katayose, Y., Starker, C., et al. (2016). A defect in 584
DNA ligase 4 enhances the frequency of TALEN-mediated targeted 585
mutagenesis in rice. Plant Physiol. 170:653-666. 586
Piatek, A., Ali, Z., Baazim, H., Li, L., Abulfaraj, A., Al-Shareef, S., Aouida, M., and 587
Mahfouz, M.M. (2015). RNA-guided transcriptional regulation in planta via 588
synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13:578-589. 589
Porebski, S., Bailey, L.G., and Baum, B.R. (1997). Modification of a CTAB DNA 590
extraction protocol for plants containing high polysaccharide and polyphenol 591
components. Plant Mol. Biol. Report. 15:8-15. 592
Ran, F.A., Cong, L., Yan, W.X., Scott, D.A., Gootenberg, J.S., Kriz, A.J., Zetsche, B., 593
Shalem, O., Wu, X., Makarova, K.S., et al. (2015). In vivo genome editing 594
using Staphylococcus aureus Cas9. Nature 520:186-191. 595
Ren, B., Yan, F., Kuang, Y., Li, N., Zhang, D., Lin, H., and Zhou, H. (2017). A 596
CRISPR/Cas9 toolkit for efficient targeted base editing to induce genetic 597
variations in rice. Sci. China Life Sci. 60:516-519. 598
Ren, B., Yan, F., Kuang, Y., Li, N., Zhang, D., Zhou, X., Lin, H., and Zhou, H. 599
(2018). Improved base editor for efficiently inducing genetic variations in 600
rice with CRISPR/Cas9-guided hyperactive hAID mutant. Mol. Plant 601
11:623-626. 602
Scheben, A., Wolter, F., Batley, J., Puchta, H., and Edwards, D. (2017). Towards 603
CRISPR/Cas crops - bringing together genomics and genome editing. New 604
Phytol. 216:682-698. 605
Schmidt C, Pacher M, and H., P. (2015). DNA break repair in plants and its 606
application for genome engineering. Methods Mol. Biol. 1864:237-266. 607
Shimatani, Z., Kashojiya, S., Takayama, M., Terada, R., Arazoe, T., Ishii, H., 608
Teramura, H., Yamamoto, T., Komatsu, H., Miura, K., et al. (2017). Targeted 609
base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase 610
fusion. Nat. Biotechnol. 35:441-443. 611
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
22
Vazquez-Vilar, M., Bernabé-Orts, J.M., Fernandez-del-Carmen, A., Ziarsolo, P., 612
Blanca, J., Granell, A., and Orzaez, D. (2016). A modular toolbox for 613
gRNA-Cas9 genome engineering in plants based on the GoldenBraid 614
standard. Plant Methods 12:10. 615
Wang, H., La Russa, M., and Qi, L.S. (2016). CRISPR/Cas9 in genome editing and 616
beyond. Annu. Rev. Biochem. 85:227-264. 617
Wang, J., Meng, X., Hu, X., Sun, T., Li, J., Wang, K., and Yu, H. (2018). xCas9 618
expands the scope of genome editing with reduced efficiency in rice. Plant 619
Biotechnol. J. Advance Access published Dec 13, 2018, 620
doi:10.1111/pbi.13053. 621
Waterworth, W.M., Drury, G.E., Bray, C.M., and West, C.E. (2011). Repairing breaks 622
in the plant genome: the importance of keeping it together. New Phytol. 623
192:805-822. 624
Wright, Addison V., Nuñez, James K., and Doudna, Jennifer A. (2016). Biology and 625
applications of CRISPR systems: harnessing nature’s toolbox for genome 626
engineering. Cell 164:29-44. 627
Yan, F., Kuang, Y., Ren, B., Wang, J., Zhang, D., Lin, H., Yang, B., Zhou, X., and 628
Zhou, H. (2018). Highly efficient A.T to G.C base editing by Cas9n-guided 629
tRNA adenosine deaminase in rice. Mol. Plant 11:631-634. 630
Yin, K., Gao, C., and Qiu, J.L. (2017). Progress and prospects in plant genome 631
editing. Nat. Plants 3:17107. 632
Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., 633
Essletzbichler, P., Volz, S.E., Joung, J., van der Oost, J., Regev, A., et al. 634
(2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas 635
system. Cell 163:759-771. 636
Zhou, H., Liu, B., Weeks, D.P., Spalding, M.H., and Yang, B. (2014). Large 637
chromosomal deletions and heritable small genetic changes induced by 638
CRISPR/Cas9 in rice. Nucleic Acids Res. 42:10903-10914. 639
Zong, Y., Wang, Y., Li, C., Zhang, R., Chen, K., Ran, Y., Qiu, J.L., Wang, D., and 640
Gao, C. (2017). Precise base editing in rice, wheat and maize with a 641
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
23
Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35:438-440.642
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
24
Figure Legends 643
Figure 1. Characterization of Cas9-NG endonuclease in rice protoplasts. 644
(A) Gene constructs of Cas9-NG, xCas9 and Cas9 used for targeted gene editing in 645
rice. 646
(B-D) Test of Cas9-NG/sgRNA activity toward NGA PAM by targeting OsCERK1 (B), 647
NGT PAM by targeting OsGSK4 (C), NGC PAM by targeting OsETR2 (D). The 648
experiments were performed at least twice, and all data are combined. The PAM 649
sequences are highlighted in green; the target sequences are in bold and the BamHI 650
recognition sites are underlined. For the Cas9-NG/sgRNA-induced gene mutations, 651
deletions and insertions are depicted as dashes and slashes/lower-case letters, 652
respectively. The number of deleted nucleotides and/or inserted nucleotides and the 653
frequency with which each DNA sequence pattern was detected are presented in the 654
columns to the right. 655
656
Figure 2. Comparison of on-target activities of Cas9-NG, xCas9 and Cas9 657
endonucleases toward different PAM sequences in rice plants. 658
(A-D) Representative mutated sequences of OsMPK11 with a NGG PAM (A), 659
OsMPK7 with a NGA PAM (B), OsMPK10 with a NGT PAM (C) and OsMPK8 with 660
a NGC PAM (D) caused by Cas9-NG/sgRNA in T0 independent transgenic lines. 661
(E) Summary of mutation frequencies induced by Cas9-NG, xCas9 and Cas9 in 662
regard to different types of PAM sequences in T0 transgenic rice plants. 663
The PAM sequences are underlined and the target sequences are in bold in the 664
wild-type sequences; nucleotide deletions and insertions are depicted as dashes and 665
lower-case letters, respectively. 666
667
Figure 3. Multiplex gene targeting by Cas9-NG endonuclease in rice plants. 668
(A) Representative mutant sequences of OsSERK1 and OsSERK2 with NGG PAMs 669
caused by Cas9-NG/sgRNA in T0 independent transgenic lines. 670
(B) Representative mutant sequences of OsGSK4 and OsETR2 with NGT PAMs 671
caused by Cas9-NG/sgRNA in T0 independent transgenic lines. 672
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
25
(C) Summary of mutation frequencies induced by Cas9-NG, xCas9 and Cas9 in T0 673
transgenic rice plants. 674
The PAM sequences are underlined and the target sequences are in bold in the 675
wild-type sequences; Deletions and insertions are depicted as dashes lower-case 676
letters, respectively. 677
678
Figure 4. Targeted base editing using Cas9-NG-fused nucleoside deaminases in 679
rice plants. 680
(A) Gene constructs of CBEs engineered with Cas9-NG, xCas9 or Cas9. 681
(B) rBE22, the Cas9-NG-based CBE, converts cytosine to thymine in the target region 682
of the endogenous OsRLCK185 gene in rice protoplasts. 683
(C) The target site of OsBZR1 gene in rice. 684
(D) Representative Sanger sequencing chromatogram of the rBE22-edited OsBZR1 685
allele in T0 transgenic line. 686
(E) Gene constructs of ABEs engineered with Cas9-NG, xCas9 or Cas9. 687
(F) rBE23, the Cas9-NG-based ABE, converts adenine to guanine in the target 688
region of the endogenous Os03g020040 gene in rice protoplasts. 689
(G) The target site of OsSERK2 gene in rice. 690
(H) Representative Sanger sequencing chromatogram of the rBE23-edited OsSERK2 691
allele in T0 transgenic line. 692
(I-J) Summary of nucleotide changes in the editing window of the endogenous 693
OsSERK2 gene caused by rBE23 (I) and rBE22 (J) in T0 independent transgenic 694
lines. 695
(K) Mutation frequencies induced by CBEs and ABEs in T0 transgenic rice plants. 696
The PAM sequences, the target sequences, the candidate bases in the putative editing 697
window and the detected nucleotide changes/the corresponding amino acids are 698
highlighted in green, bold, red and blue, respectively. For (B) and (F), the restriction 699
enzyme-cutting sites are underlined. For (D) and (H), the nucleotide changes are 700
underlined in the sequencing chromatograms. 701
702
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
26
Figure 5. Transcriptional activation of target genes mediated by 703
dCas9-NG-TAD in rice plants. 704
(A) Gene constructs of dCas9-NG- and dCas9-based transcriptional activators. 705
(B) Diagram of the OsCOI2 gene structure and the target sites of gRNAs in the 706
promoter region. 707
(C) qRT-PCR analysis of the OsCOI2 transcript levels in T0 independent transgenic 708
lines. 709
(D) Diagram of the OsSERK2 gene structure and the target sites of gRNAs in the 710
promoter region. 711
(E) qRT-PCR analysis of the OsSERK2 transcript levels in T0 independent 712
transgenic lines. 713
For (B) and (D), the PAM sequences and the target sequences on the sense and 714
antisense DNA strands are highlighted in green and bold, respectively. For (C) and 715
(E), the relative expression of the targeted genes was normalized to the amount of 716
OsActin cDNA in each transgenic line, fold-changes were then determined using 717
transgenic plants that express dCas9-TAD alone as a control. The data shown here 718
are representatives of four independent experiments. 719
720
Figure 6. Cas9-NG recognizes novel atypical PAM sequences other than NG in 721
rice. 722
(A-D) Sequence mutations of OsGSK4 at the GAC PAM site (A), OsGSK4 at the TTG 723
PAM site (B), Os03g02040 at the GTT PAM site (C), and OsCERK1 at the GCG 724
PAM site (D) caused by Cas9-NG/sgRNA in T0 independent transgenic lines. The 725
atypical PAM sequences are underlined; the target sequences in bold in the wild-type 726
sequences and nucleotide deletions are depicted as dashes. 727
(E) Summary of the mutation frequencies induced by Cas9-NG in regard to each 728
atypical PAM sequence in T0 transgenic rice plants. 729
Top Related