Table 1 Optimization of the CRISPR-Cas system in plants

From: CRISPR technology is revolutionizing the improvement of tomato and other fruit crops

Name

From

Function

Crop species

Refs.

Cas proteins

 St1Cas9

Streptococcus thermophilus

Size is smaller; recognizes longer PAMs (“NNAGAA” or “NNGGAA”)

Arabidopsis

45

 SaCas9

Staphylococcus aureus

Size is smaller; recognizes longer PAMs (“NNGGGT” or “NNGAA”)

Arabidopsis; tobacco

45,46

 SpCas9-VQR

Streptococcus pyogenes

Recognizes “NGA” PAM

Rice

47

 SpCas9- VRER

Streptococcus pyogenes

Recognizes “NGCG” PAM

Rice

47

 Cas12a (Cpf1)

Acidaminococcus sp. BV3L6 (As); Francisellanovicida (Fn); Lachnospiraceae bacterium ND2006 (Lb)

Recognizes “TTTN” or “TTN” PAMs; targets DNA to introduce a 5′ overhang; guided by a shorter crRNA; exhibits little off-target activity

Arabidopsis; maize; rice; soybean; tobacco

48,49,50,51

 Cas13a (C2c2)

Leptotrichiashahii

Targets single-stranded RNA with PFS of A, U, or C

Rice; tobacco

52,53

 nCas9

Streptococcus pyogenes

Cas9 nickase contains a mutation in either of the two nuclease domains of Cas9 protein. It induces SSBs

Arabidopsis; rice; tomato

54,55,56

 dCas9

Streptococcus pyogenes

Deficient Cas9 contains mutations in both nuclease domains of Cas9 protein. without cleavage activity. The dCas9-based regulator can be developed when fused with transcriptional activators or repressors

Arabidopsis; maize; rice; tobacco; wheat

56,57,58,59

Promoters

 

Preferential expression

Crop species

Refs.

Cas promoters

 YAO

 

Tissues undergoing active cell division including the shoot apical and root meristem, embryo sac, embryo, endosperm, and pollen

Arabidopsis; citrus

60,61

 SPL

 

Sporogenous cells and microsporocytes

Arabidopsis

62

 EC1.1/EC1.2

 

Egg cells and one-cell stage embryos

Arabidopsis

63,64

 ICU2

 

Meristematic regions

Arabidopsis

65

 EF1α, hisH4

 

Meristematic and reproductive tissues

Arabidopsis

66

 MGE

 

Meiosis stage

Arabidopsis

67

 DMC1

 

Meiocytes

Arabidopsis; maize

68,69

 RPS5A

 

At all developmental stages

Arabidopsis

70

Strategy

  

Crop species

Refs.

sgRNAs

 Assemble multiple sgRNA expression cassettes into CRISPR-Cas vector

Arabidopsis; maize; Populus; rice; tobacco; tomato

71,72,73,74,75

 Produce numerous sgRNAs from a single polycistronic gene via the endogenous tRNA-processing system

Maize; potato; rice; tomato; wheat

76,77,78,79,80

  1. PAM protospacer adjacent motif, sgRNA single-guide RNA, CRISPR-Cas clustered regularly interspaced short palindromic repeat-CRISPR-associated, tRNA transfer RNA, PFS protospacer flanking sequence, SSBs single-strand breaks, crRNA CRISPR RNA
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