Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

Meng, Xiangbing; Hu, Xixun; Liu, Qing; Song, Xiaoguang; Gao, Caixia; Li, Jiayang; Wang, Kejian

doi:10.1007/s11427-017-9247-9

Cited by 55 publications

(37 citation statements)

References 5 publications

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“…Among the four NNG PAMs assessed, xCas9 3.6 and 3.7 exhibited limited ability (4.17%) to edit three NGG PAM sites (Figure 1c). In addition to NGG PAM sites, SpCas9 also effectively introduced mutations at NAG PAM sites (2.08%-60.42%), which is consistent with previous report (Meng et al, 2018).…”

supporting

confidence: 91%

“…In an effort to overcome this limitation, several studies have reported that other Cas effectors (Cpf1 for AT-rich PAMs) and engineered Cas9 variants (VQR for NGA PAMs and VRER for NGCG PAMs) could be employed with other PAMs for rice genome editing (Hu et al, 2016;Zetsche et al, 2015). In addition, another study showed that SpCas9 could robustly recognize NAG PAMs and cleave target sites in rice genome (Meng et al, 2018). Although several tools have been developed for genome editing, versatile nucleases recognizing various PAMs are still largely required to expand the genome editing toolbox.…”

mentioning

confidence: 99%

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xCas9 expands the scope of genome editing with reduced efficiency in rice

Wang

Meng

et al. 2019

Plant Biotechnology Journal

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supporting

confidence: 91%

mentioning

confidence: 99%

xCas9 expands the scope of genome editing with reduced efficiency in rice

Wang

Meng

et al. 2019

Plant Biotechnology Journal

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“…Previous studies have shown that the wild type SpCas9 protein can recognize not only the canonical NGG PAM but also the non-canonical NAG PAM sequence in the rice genome and exhibits robust editing efficiencies at some sites with NAG PAMs (Meng et al, 2018). We wondered whether our ABE-P1 (adenine base editor plant version 1) vector with a wild type SpCas9 (D10A) nickase (Hua et al, 2018) can also edit genome sequences with NAG PAMs.…”

Section: Resultsmentioning

confidence: 99%

Expanding the base editing scope in rice by using Cas9 variants

Hua

Tao

Zhu

2018

Plant Biotechnology Journal

179

137

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Base editing is a novel genome editing strategy that enables irreversible base conversion at target loci without the need for double stranded break induction or homology-directed repair. Here, we developed new adenine and cytosine base editors with engineered SpCas9 and SaCas9 variants that substantially expand the targetable sites in the rice genome. These new base editors can edit endogenous genes in the rice genome with various efficiencies. Moreover, we show that adenine and cytosine base editing can be simultaneously executed in rice. The new base editors described here will be useful in rice functional genomics research and will advance precision molecular breeding in crops.

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“…4c ). Further analysis these 12 off-target sites found four have silent mutations in NGG PAM and one has a non-canonical CAG PAM, which was reported as an alternative PAM (NAG) for SpCas9 nuclease 32 and recently shown to mediate Cas9 activity in rice 33 . All mutations at these 12 sites were indels, and, importantly, the two Cas9-J T0 lines carried distinct alleles at these sites ( Fig.…”

Section: Resultsmentioning

confidence: 93%

A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 nucleases in rice

Tang

Liu

Zhou

et al. 2018

Preprint

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33Targeting specificity has been an essential issue for applying genome editing 34 systems in functional genomics, precise medicine and plant breeding. 35 Understanding the scope of off-target mutations in Cas9 or Cpf1-edited crops is 36 critical for research and regulation. In plants, only limited studies had used whole-37 genome sequencing (WGS) to test off-target effects of Cas9. However, the cause 38 of numerous discovered mutations is still controversial. Furthermore, WGS based 39 off-target analysis of Cpf1 has not been reported in any higher organism to date. 40 Here, we conducted a WGS analysis of 34 plants edited by Cas9 and 15 plants 41 edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice, 42 a major food crop with a genome size of ~380 Mb. The sequencing depth ranged 43 from 45X to 105X with reads mapping rate above 96%. Our results clearly show that 44 most mutations in edited plants were created by tissue culture process, which 45 caused ~102 to 148 single nucleotide variations (SNVs) and ~32 to 83 46 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) 47 and 3 Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA 48 resulted in off-target mutations in T0 lines at sites predicted by computer programs. 49 Moreover, we cannot find evidence for bona fide off-target mutations due to 50 continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation. Taken 51 together, our comprehensive and rigorous analysis of WGS big data across 52 multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in 53 generating targeted DNA modifications and off-targeting can be avoided by 54 designing guide RNAs with high specificity. 55 Bacterial type II CRISPR-Cas9 systems can effectively induce RNA-guided DNA 56 double strand breaks (DSBs) 1 , making them popular tools for genome editing in bacteria 2 , 57 animal cells 3 , mammalian systems 4-7 and plants 8-11 . The most widely used Streptococcus 58 3 pyogenes Cas9 (SpCas9) uses ~20 nucleotides (nt) of a single guide RNA (sgRNA) to 59 recognize a complementary target DNA site along with an NGG protospacer adjacent 60 motif (PAM) 1, 12 . More recently, type V CRISPR-Cpf1 was shown to mediate efficient 61 genome editing in human cells 13 and plants 14, 15 . Cpf1 uses ~23 nt of an RNA guide to 62 target DNA with a TTTV PAM 13 . RNA-guided nucleases (RGNs) such as Cas9 and Cpf1 63 represent versatile genome editing tools that promise to advance basic science, enable 64 personalized medicine and accelerate crop breeding. However, Cas9 may cause 65 undesired off-target mutations due to sgRNAs recognizing DNA sequences with one to a 66 few nucleotide mismatches; albeit with reduced nuclease binding and cleavage activity 1, 67 6, 16, 17 . Although similar rules apply to Cpf1, recent studies in human cells 18, 19 have shown 68 Cpf1 is generally more specific than Cas9. 69 Understanding the scope of off-target mutations in Cas9 or Cpf1-edited crops is 70 critical for research and reg...

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Robust genome editing of CRISPR-Cas9 at NAG PAMs in rice

Cited by 55 publications

References 5 publications

xCas9 expands the scope of genome editing with reduced efficiency in rice

xCas9 expands the scope of genome editing with reduced efficiency in rice

Expanding the base editing scope in rice by using Cas9 variants

A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 nucleases in rice

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