Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

Abstract

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum

“…edited both gene copies in all four, whereas AccI gRNA edited both gene copies in only two of the four. This confirmed the better efficiency of the BsrGI gRNA than AccI gRNA in Brachypodium editing and may be in part explained by different cleavage efficiencies of various gRNAs (Bruegmann et al, 2019). It is also consistent with the results obtained using the transient protoplast assay, as positive results were obtained for BsrGI gRNA and not for AccI gRNA.…”

CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

“…Optimizing guide designs can also reduce the frequency of OTEs ( 31 ). Many features in an sgRNA determine specificity including the seed sequence (a 10–12 bp region proximal to PAM on 3′ of spacer sequence) ( 29 , 53 ), GC content ( 54 , 55 ), and modifications such as 5′ truncation of the sgRNA ( 56 ). Several platforms have also been designed to provide optimized guide sequences against target genes, including E-Crisp ( 31 , 57 ), CRISPR-design, CasOFFinder, and others ( 31 ).…”

A general onepot-method for nucleic acid detection with CRISPR-Cas12a

“…Cleavage e ciency of CRISPR/Cas9 systems seemed to be in uenced by GC content and purine residues in the gRNA end 22 . We were therefore interested in knowing whether the trans activity of Cas12a was dependent on the sequence of reporters.…”