Single Base Editing with CRISPR in Plant Genetic Modification

Due to the fact that HDR (homology directed repair) occurs at a relatively low frequency especially in nondividing cells compared to NHEJ (non-homologous end joining), scientists have developed advanced CRISPR/Cas9 systems called CRISPR base editors that induce single nucleotide change at target loci without the need for double stranded break formation or a donor DNA template provision. Lifeasible has applied them successfully in monocots and dicots such as wheat, rice, maize, Arabidopsis, and tomato.

CRISPR base editors contain catalytically inactive dCas9 (dead Cas 9) or nCas9 (Cas9 nickase) and a cytidine deaminase or adenine deaminase. The CBEs (cytidine base editors) can convert cytosine to uracil, which is subsequently converted to thymine through DNA replication or repair. Similarly, ABEs (adenine base editors) mediate the conversion of adenine to inosine, and then subsequently is corrected to guanine, thereby converting A into G.

Advantages of CRISPR base editors are as follows：

• Simple construction: the system can realize precise base editing without providing a DNA template.
• Many advanced systems acquired for more effective and efficient editing.
• Many Cas9 variants such as SpCas9, SaCas9, SpVQR-Cas9, SpEQR-Cas9, SpVRER-Cas9, and SaKKH-Cas9 with different PAM (protospacer-adjacent motif) specificities.
• Mutant cytidine deaminase domains that exhibit narrowed editing windows and adenine deaminase (e.g., ABE7.10) with high product purity and low off-target editing efficiency.
• TAM and CRISPR-X systems which can convert C into A, G, or T and G into A, C, or T.
• CRISPR/Cas13 system editing mRNA sequences and transforming adenosine to inosine via the deaminase activity of ADAR2.

Figure 1. Structural representation of cytidine (left) and adenine (right) base editors (Eid et al., 2018)

Lifeasible offers our worldwide customers a well-established one-stop service package, including vector constructions, transformations and subsequently gene function analysis. With valuable national and international tie-ups with renowned companies, we can provide innovative solutions in gene editing and transgenic skills.

Experimental process: