Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering

Abstract

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 from Staphylococcus aureus (SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

“…In addition, protein-engineering approaches resulted in altered SpCas9 variants with potentially reduced off-target activity. Several SpCas9-variants like SpCas9-HF, eSpCas9 (1.0), or eSpCas9 (1.1) have been engineered and were already applied in plants (Zhang et al, 2017;Raitskin et al, 2019). These nuclease variants were described to nearly entirely avoid off-targeting (Tycko et al, 2016).…”

A modular cloning toolkit for genome editing in plants

“…Benson Hill (USA) is the source of the SuCms1 vector/sequence and the license to the licensed patent rights. pFH22: The xCas9-3.7 gene was amplified from the vector pEPOR0SP0011 (Raitskin et al 2019) using primers FH407/FH407. The gel-purified PCR product was assembled into pICH41308 (Weber et al 2011) (Addgene ID #47998) using a GG cut-ligation reaction via BpiI.…”

Metabolomics should be deployed in the identification and characterization of gene‐edited crops

“…Although the technique has been much reviewed in recent years (Baltes and Voytas, ; Puchta, ; Scheben et al ., ; Komor et al ., ; Kumlehn et al ., ; Scheben and Edwards, ), it is imperative to realise that our understanding of both its scope and the mechanisms by which it can operate are rapidly evolving. For example, targeting precision has recently been improved in plants and other organisms by a range of means, including the use of different CRISPR‐associated nucleases (Komor et al ., ; Kim et al ., ; Raitskin et al ., ), in addition to now achieving editing without the need for double‐stranded DNA breaks (Gaudelli et al ., ). Enhanced precision should reduce so‐called ‘off‐target’ effects, whereby genes other than that targeted gene are edited.…”