Chandran Pfitzner

• Bachelor of Science (Molecular Biology)
• PostDoc Position at Macquarie University

CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable mu...

Self-replicating gene drives that can spread deleterious alleles through animal populations have been promoted as a much needed but controversial ‘silver bullet’ for controlling invasive alien species. Homing-based drives comprise an endonuclease and a guide RNA (gRNA) that are replicated during meiosis via homologous recombination. However, their...

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drives have been developed in insects and yeast but not in mammals. Here we investigated the efficiency of CRISPR/Cas9-based gene drives in Mus musculus by...

Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols...

Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols...

Non-clustered protocadherins (ncPcdhs) are adhesive molecules with spatio-temporally regulated overlapping expression in the developing nervous system. Although their unique role in neurogenesis has been widely studied, their combinatorial role in brain physiology and pathology is poorly understood. Using probabilistic cell typing by in situ sequen...

Gene drives are genetic elements that are transmitted to greater than 50% of offspring and have potential for population modification or suppression. While gene drives are known to occur naturally, the recent emergence of CRISPR-Cas9 genome-editing technology has enabled generation of synthetic gene drives in a range of organisms including mosquito...

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drive systems have been developed in insects and yeast but not in mammals. Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in Mus musc...

Paired-nickase DSB induction by pDG462. Extended figures of Fig 3 with more independent samples. Smaller bands produced after T7E1 digestion (blue arrows) indicated presence of mutation in samples treated with paired-nickase pDG462 Sox1A/Sox1B (A) or Sox3A/Sox3B (B). Each sample came from independent transfections. (TIF)

Efficient dual cutting mediated by pDG459 vector. Extended figures of Fig 2B with more independent samples. (A) BfuAI and SacI RFLP analyses indicated efficient dual cuts from pDG459 Sox1A/Sox3A. (B) ApaI and SfoI RFLP analyses indicated efficient dual cuts from pDG459 Sox1B/Sox3B. WT products after digestions (red arrows) were absent in pDG459-tre...

Mutation inductions mediated by vectors pDG330 and pDG458. (A) Transfection of pDG330 Sox1A/Sox3A into mouse ES cells induced mutations at both targets which were indicated by smaller fragments after T7E1 assay (blue arrows). (B) BfuAI and SacI RFLP were used to assess the mutation induction in Sox1A and Sox3A sites, respectively, after treatment o...

Paired-nickase-mediated mutation inductions by pDG335 and pDG461 vectors. T7E1 assay showed that expression of paired-nickase gRNAs Sox3A/Sox3B from pDG335 (A) or pDG461 (B) induced mutations in the Sox3 locus as indicated by the presence of cut products (blue arrows). Each sample came from independent transfections. (TIF)