This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments.


Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli

Relative chromosome dosage, i.e., increases or decreases in the number of copies of specific chromosome regions in one sample versus another, can be determined using aligned read-counts from Illumina sequencing (Henry et al., 2010). The following protocol was used to identify the different classes of aneuploids that result from uniparental genome elimination in Arabidopsis thaliana, including chromosomes that have undergone chromothripsis (Tan et al., 2015). Uniparental genome elimination results in the production of haploid progeny from crosses to specific strains called “haploid inducers” (Ravi et al., 2014). On the other hand, chromothripsis, which was first discovered in cancer genomes, is a phenomenon that results in clustered, highly rearranged chromosomes. In plants, chromothripsis has been observed as a result of genome elimination (Tan et al., 2015). Detecting variation in chromosome dosage has multiple applications beside those linked to genome elimination. For example, a dosage variant population of poplar hybrids was created by gamma-irradiation of pollen grains. Hundreds of dosage lesions, insertions and deletions, were identified using this technique and provide a way to associate loci with the phenotypic consequences observed in this population (Henry et al., 2015).

This method has been successfully used to detect changes in chromosome dosage in many different species, including Arabidopsis thaliana (Tan et al., 2015), Arabidopsis suecica (Ravi et al., 2014), rice (Henry et al., 2010) and poplar (Henry et al., 2015). It is important to note that dosage plots always indicate dosage variation relative to the control sample used (Note 1). Therefore, this approach is not suitable to detect ploidy variants (diploid vs triploid, for example). Similarly, this technique does not allow the detection of balanced chromosomal rearrangements such as reciprocal translocations.