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Custom-designed nucleases, including CRISPR-Cas9 and CRISPR-Cpf1, are widely used to realize the precise genome editing. The high-coverage, low-cost and quantifiability make high-throughput sequencing (NGS) to be an effective method to assess the efficiency of custom-designed nucleases. However, contrast to standardized transcriptome protocol, the NGS data lacks a user-friendly pipeline connecting different tools that can automatically calculate mutation, evaluate editing efficiency and realize in a more comprehensive dataset that can be visualized. Here, we have developed an automatic stand-alone toolkit based on python script, namely CRISPRMatch, to process the high-throughput genome-editing data of CRISPR nuclease transformed protoplasts by integrating analysis steps like mapping reads and normalizing reads count, calculating mutation frequency (deletion and insertion), evaluating efficiency and accuracy of genome-editing, and visualizing the results (tables and figures). Both of CRISPR-Cas9 and CRISPR-Cpf1 nucleases are supported by CRISPRMatch toolkit and the integrated code has been released on GitHub (https://github.com/zhangtaolab/CRISPRMatch).

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Two photoactivatable near infrared fluorescent proteins (NIR FPs) named “PAiRFP1” and “PAiRFP2” are formed by directed molecular evolution from Agp2, a bathy bacteriophytochrome of Agrobacterium tumefaciens C58. There are 15 and 24 amino acid substitutions in the structure of PAiRFP1 and PAiRFP2, respectively. A comprehensive molecular exploration of these bacteriophytochrome photoreceptors (BphPs) are required to understand the structure dynamics. In this study, the NIR fluorescence emission spectra for PAiRFP1 were recorded upon repeated excitation and the fluorescence intensity of PAiRFP1 tends to increase as the irradiation time was prolonged. We also predicted that mutations Q168L, V244F, and A480V in Agp2 will enhance the molecular stability and flexibility. During molecular dynamics (MD) simulations, the average root mean square deviations of Agp2, PAiRFP1, and PAiRFP2 were found to be 0.40, 0.49, and 0.48 nm, respectively. The structure of PAiRFP1 and PAiRFP2 were more deviated than Agp2 from its native conformation and the hydrophobic regions that were buried in PAiRFP1 and PAiRFP2 core exposed to solvent molecules. The eigenvalues and the trace of covariance matrix were found to be high for PAiRFP1 (597.90 nm2) and PAiRFP2 (726.74 nm2) when compared with Agp2 (535.79 nm2). It was also found that PAiRFP1 has more sharp Gibbs free energy global minima than Agp2 and PAiRFP2. This comparative analysis will help to gain deeper understanding on the structural changes during the evolution of photoactivatable NIR FPs. Further work can be carried out by combining PCR-based directed mutagenesis and spectroscopic methods to provide strategies for the rational designing of these PAiRFPs.