Construction and Activity Verification of Ribonucleoprotein Complex for Gene Editing

doi:10.13560/j.cnki.biotech.bull.1985.2021-1353

Abstract

Abstract:

CRISPR/Cas9 gene editing technology has the advantages of simple operation,low cost and high editing efficiency,and has been widely used in the research of gene editing in animals,plants and microorganisms. In this technical system,ribonucleoprotein（RNP）complex assembled from Cas9 protein and guide RNA（gRNA）,exerts the function of nucleic acid cleavage. At present,there are more and more researches on direct delivery of RNP complex assembled in vitro into recipient cells for gene editing,which has become an effective means to improve editing efficiency,and reduce off-target rate. The purpose of this study is to obtain an RNP complex with high cleavage activity. In this research, the Cas9 protein was successfully expressed in Escherichia coli BL21（DE3）,and the recombinant Cas9 protein was purified by His-tag affinity resin. At the same time,the T7 transcription kit in vitro transcription and purification of gRNA was performed,so that the purified Cas9 protein and gRNA spontaneously assembled into RNP complexes. After in vitro activity detection,RNP complexes demonstrated good DNA double-stranded shearing activity;and RNP was co-transformed the Aspergillus niger strain with donor DNA,the target gene was successfully knocked out,and the editing efficiency reached more than 90%. The RNP complex obtained in this study can be applied to gene editing of filamentous fungi,saving time and cost,and promoting the application of RNP-mediated gene editing technology in more fields.

Key words: Cas9 protein, RNP complex, Escherichia coli, activity detection

GAO Wei-xin, HUANG Huo-qing, ZHAO Jing, ZHANG Xin, YANG Ning, YANG Hao-meng. Construction and Activity Verification of Ribonucleoprotein Complex for Gene Editing[J]. Biotechnology Bulletin, 2022, 38(8): 60-68.

Figures/Tables 8

Table 1 Primer names and sequence information

Primer name	Primer sequence（5'-3'）
Pet-Insert-F	taagaaggagatataccatggATAAGAAATACTCAATAGGCTTAGA
Pet-Insert-R	gtggtggtggtggtgctcgagGACCTTGCGCTTCTTCTTGGgagggtcacctcctagctgactcaaatc
glucoamylase-F	atgtcgttccgatctctactcgc
glucoamylase-R	ctaccgccaggtgtcagtcac
223up-F	atgtcgttccgatctctactcgccctg
223up-R	acttcttcccagagatctgcgatcg
223down-F	caatggctcgtctttctttacgattgc
223down-R	aggtgacagtcacactgctgtagg
hph-F	gcagatctctgggaagaagtgacagaagatgatattgaaggagcac
hph-R	aaagaaagacgagccattggtatctggaagaggtaaacccgaaacg
hph-jc-F	ctcgccgatagtggaaaccgac
hph-jc-R	cgcaccaagttatcgtgcacc
GA223homo-up-jc-F	cccacaccatcggagattcgtcgcc

Table 2 Sequence information of in vitro transcription templates of RNA

Name	Sequence（5'-3'）
gRNA	TAATACGACTCACTATAGGgatctctgggaagaagtcaa GTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT
cgRNA	TAATACGACTCACTATAGGgatgggattcgtgagtacaaGTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT

Table 3 Reaction system of test group for RNP complex activity verification

	添加量Additive amount
	1	2	3	4	5	6
Cas9	200 ng	400 ng	800 ng	1.6 μg	3.2 μg	6.4 μg
gRNA	200 ng	400 ng	800 ng	1.6 μg	3.2 μg	6.4 μg
DNA	200 ng	200 ng	200 ng	200 ng	200 ng	200 ng

Fig.1 SDA-PAGE analysis of recombinant Cas9 M：Protein molecular mass marker;1：disrupted cell supernatant after induction;2：purified Cas9

Fig.2 In vitro activity verification of RNP complex A: Negative control. B: Experimental group, 1-6（①-⑥）respectively indicates the addition amount of cCas9（Cas9）,gRNA,and DNA at different concentrations. The addition amount of the three is shown in Table 3. M：DNA molecular mass marker

Fig.3 Schematic diagram of RNP complex editing process in vivo

Fig.4 Verification of positive transformants by PCR M：DNA molecular mass marker;1-11 are different positive transformants;CK is the original strain as control

Fig.5 SDS-PAGE analysis of supernatant of transformant fermentation broth M：Protein molecular mass marker;1-11 are the fermentation broth supernatants of different transformants;CK is the original strain as control

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