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Optimization of CRISPR/Cas9 Genome Editing Systems in Protoplasts of Setaria italica

LIU Guangyu, XU Xiaojing, XIA Keke, SUN Haixi, TAO Yueru, CUI Zhen, GU Ying

Journal of Henan Agricultural Sciences 2022, 51 (1): 34-42. DOI: 10.15933/j.cnki.1004-3268.2022.01.005

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To optimize and screen efficient genome editing systems in millet（Setaria italica），multiple CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISRP‐associated nuclease 9）genome editing systems were constructed with six kinds of gRNAs designed for phytoene desaturase（SiPDS） gene（gRNA1—gRNA2 for exon 1，gRNA3—gRNA6 for exon 12），and transformed into protoplasts of millet by polyethylene glycol‐mediated method. Then the mutation efficiency against SiPDS gene was rapidly detected by large‐scale parallel sequencing. The results showed that the protoplast transformation system established by the young stems of 7 day‐old millet etiolated seedlings had high transformation efficiency from 50. 44% to 57. 36%. Using this method，the gene editing systems with Cas9 gene driven by Super and Ubi promoters were transferred into millet protoplasts，the mutation efficiencies of SiPDS gene were 0. 5% and 5. 5% respectively. Thus，the Cas9 gene of gene editing systems with single gRNA，double gRNA or tRNA cassettes were driven by Ubi promoter，and mutation efficiency of SiPDS gene induced by these systems was compared. It was found that the mutation efficiencies of gene editing system with double gRNA cassettes such as Ubi‐dgRNAE1 and Ubi‐dgRNAE12 were 1. 66 and 1. 11 times higher than that with single gRNA system. The mutation efficiency induced by tRNA‐based system Ubi‐tRNA was 51. 24%，which was 5. 87 times higher than that with single gRNA system. Moreover，Ubi‐tRNA could simultaneously edit multiple sites of SiPDS gene，the multi‐site mutation frequency was 3. 23%. The cleavage activities in vitro of ribonucleoprotein（RNP）complexes which were mixture of gRNA3，gRNA4 or gRNA5 transcripts with Cas9 protein respectively were compared，it was found that RNP‐gRNA5 complex had the highest cleavage activity in vitro，the mutation efficiency of SiPDS gene was 2. 0%，and the dominant mutation type was deletion less than 3 bp.

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Application Progress of CRISPR/Cas9 Technology in Crop Genetic Breeding

JIAO Yaolei, WANG Chunsheng, QU Shuo, SUN Shanshan, ZHU Tingting, ZHAO He, WANG Piwu

Journal of Henan Agricultural Sciences 2021, 50 (7): 1-7. DOI: 10.15933/j.cnki.1004-3268.2021.07.001

Abstract （737）

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CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISRP‑associated nuclease 9）is a new type of genome⁃directed editing technology after ZFNs（zinc finger nucleases）and TALENs（transcription activator like effector nucleases）.Compared with the previous two generations of technology，it has the characteristics of simplicity and high efficiency.CRISPR/Cas9 is not only a basic research tool，but also has become one of currently useful molecular breeding tools，and important progress has been made in crop genetic improvement. The structure，classification，action mechanism of the CRISPR/Cas9 system and its application progress in crop quality improvement，yield enhancement， resistance breeding and male sterile material selection were reviewed，its existing problems were discussed，and its application prospects were prospected.

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Optimization of Identification of Gene Editing Progeny by CRISPR-Cas9 System in Arabidopsis thaliana

LI Ziwen, LIU Yan, LÜ Mengyang, GAO Kaili, ZHANG Hairong

Journal of Henan Agricultural Sciences 2021, 50 (4): 17-21. DOI: 10.15933/j.cnki.1004-3268.2021.04.003

Abstract （327）

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Nowadays, the technology for site-specific editing of plants using the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system has been matured,but the identification of the progeny of the edited plant is still time-consuming and labor-intensive.In order to optimize the identification method,we took Arabidopsis sugar-dependent 1( SDP1)gene as the editing object.Sequence analysis showed that there was protospacer adjacent motif(PAM)in exons of SDP1 gene,and there were four suitable restriction sites in upstream of PAM sequences.We screened these restriction sites for their positions and performance,selected the restriction site Sac Ⅰ in the second exon of SDP1 gene as the editing target,and constructed the CRISPR- SDP1-Cas9 vector.Through enzyme digestion identification and sequencing analysis of T1 seedlings screened with hygromycin,the heterozygous edited lines with three bands were identified.Among 71 randomly selected T1 plants,24 heterozygous edited lines were found,the editing efficiency was 16.9%.Among 48 T2 plants,5 homozygous edited lines were screened out,and the editing sites were all insertions at the Sac Ⅰ restriction site.In summary, the appropriate restriction sites near the PAM sequence of the gene to be edited were selected as editing targets,and the progeny plants were identified through the restriction enzyme digestion and sequencing analysis.The traditional method identifing the progeny of CRISPR-Cas9 transgenic plants is extracting DNA from the progeny plants,amplifing the target site by PCR and connecting it to the T vector for sequencing several times.This method is simple,fast,economical and practical compared with the traditional method,providing a simple and quick method for CRISPR-Cas9 gene-edited progeny identification.

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Expression and Purification of CRISPR/Cas13a and Establishment of Its Collateral RNase Activity Assay

WANG Xun, ZHANG Yuhang, SUN Yaning, LI Qingmei, LI Ge, WANG Li, GUO Junqing, DENG Ruiguang, ZHANG Gaiping

Journal of Henan Agricultural Sciences 2021, 50 (4): 147-153. DOI: 10.15933/j.cnki.1004-3268.2021.04.019

Abstract （372）

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To estabish LwaCas13a molecular detection platform,LwaCas13a was expressed in E.coli and its collateral RNase activity was determined.The recombinant plasmid pET His6-TwinStrep-SUMOLwaCas13a was transformed into Rosetta(DE3) competent cells to induce expression.The results of SDSPAGE electrophoresis and Western blot analysis showed that the soluble LwaCas13a recombinant protein was successfully expressed. Optimization of inducing temperature and time showed that maximal production of LwaCas13a was obtained at 15 h after induction at 16℃ .The expressed LwaCas13a recombinant protein was purified by nickel column affinity chromatography to obtain a single target band with ideal purification effect.In addition,the collateral RNase assay was also established with in vitro synthesized CRISPR RNA and target RNA.The assay could be performed within 30 min at constant 37℃ ,with the detection limit of 31.2 nmol/L.To sum up,the LwaCas13a was successfully expressed in E.coli Rosetta (DE3).Purified LwaCas13a exhibited ideal collateral RNase activity,and the collateral RNase assay for LwaCas13a was optimized and established.

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