MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems.
Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Hai-Yan
HY
Zhang
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Xing-Hui
XH
Wang
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Li
L
Dong
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Zhi-Ping
ZP
Wang
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Bing
B
Liu
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Jie
J
Lv
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Hui-Li
HL
Xing
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Chun-Yan
CY
Han
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Xue-Chen
XC
Wang
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Qi-Jun
QJ
Chen
D017360:Arabidopsis / Q000235:genetics; D064113:CRISPR-Cas Systems; D004926:Escherichia coli; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*