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.

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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: hbzhou@ippcaas.cn; Phone: 17

86-10-62815914; Fax: 86-10-62894642 or Honghui Lin; Email: hhlin@scu.edu.cn; 18

Phone: 86-28-85411792; Fax: 86-28-85415300 19

20

Running title: Targeted genome engineering in rice with Cas9-NG 21

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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(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

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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

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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

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(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

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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