Abstract
Nucleases used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. So far CRISPR-Cas, the RNA-guided nucleases, is the most advanced genome engineering system. The CRISPR nucleases allows recognition of a particular genomic sequence with two distinct molecular interactions: first, by direct interaction between the nuclease and the protospacer-adjacent motif, wherein discrete amino acids interact with DNA base pairs; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report the application of the single strand annealing cellular assay to analyze and quantify nuclease activity of wild type and mutant CRISPR-Cpf1. Using this heterologous marker system based on GFP activity, we observed a comparable PAM recognition selectivity with the NGS analysis. The heterologous marker system has revealed that LbCpf1 is a more specific nuclease than AsCpf1 in a cellular context. We controlled the in vitro activity of the Cpf1 nuclease complexes expressed in mammalian cells and demonstrated that they are responsible of the DNA cleavage at the target site. In addition, we generated and tested LbCpf1 variants with several combinations of mutations at the PAM-recognition positions G532, K538 and Y542. Finally, we showed that the results of the in vitro DNA cleavage assay with the wild type and mutants LbCpf1 corroborate with the selection of 6TG resistant cells associated to the genomic disruption of hprt gene.
Highlights
For reverse genetics approaches, genomic modification is often required to establish the functional link between an observed phenotype and a particular gene
We first investigated the Clustered regularly interspaced short palindromic repeat (CRISPR) from Prevotella and Francisella 1 (Cpf1) proteins Protospacer Adjacent Motif (PAM) selectivity in a cellular context by analyzing the activity of several CRISPR RNA (crRNA) with different PAM sequences that target the same genomic area corresponding to the hprt exon 3
The crRNA targeting the site flanked by the PAM 5 -TTTa-3 sequence was used as a control, to verify that the activity of AsCpf1 and LbCpf1 are similar under our experimental conditions
Summary
Genomic modification is often required to establish the functional link between an observed phenotype and a particular gene. Genetically modified cells or organisms can be isolated from the population upon a positive selection pressure. Disruption of the hprt gene allows a survival of the mutated cells upon treatment with the cytotoxic chemical agent 6-ThioGuanine (6-TG) (Fenwick and Caskey, 1975), leading to positive selection of hprt homozygote -/- cells in cultures
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