Selected Grantee Publications
Endogenous Zebrafish Neural Cre Drivers Generated by CRISPR/Cas9 Short Homology Directed Targeted Integration
Almeida et al., Scientific Reports. 2021.
https://pubmed.ncbi.nlm.nih.gov/33462297/
Almeida et al. previously reported precision targeted integration of reporter DNA in zebrafish using CRISPR/Cas9. Here, they isolated zebrafish Cre recombinase drivers. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. They observed high rates of germline transmission from 10 to 100% (10% olig2; 20% neurod1; 100% ascl1b). The lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the cell populations. Results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of targeted genes. This approach is a cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers. Supported by ORIP (R24OD020166).
Deploying MMEJ using MENdel in Precision Gene Editing Applications for Gene Therapy and Functional Genomics
Martínez-Gálvez et al., Nucleic Acids Research. 2021.
https://academic.oup.com/nar/article/49/1/67/6030233
Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Martinez-Galvez et al. compared three DSB repair prediction algorithms - MENTHU, inDelphi, and Lindel. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. The investigators report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations. They suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings to be viable for nearly all loss-of-function based gene editing therapeutic applications. Supported by ORIP (R24OD020166) and NIGMS.
Antibody-Mediated Depletion of Viral Reservoirs is Limited in SIV-Infected Macaques Treated Early With Antiretroviral Therapy
Swanstrom et al., Journal of Clinical Investigation. 2021.
https://doi.org/10.1172/JCI142421
Virus-specific strategies to target the latent HIV reservoir in individuals on combination antiretroviral therapy (cART) have been limited by inefficient induction of viral protein expression. Researchers used rhesus macaques to investigate an antibody-mediated reservoir targeting strategy, targeting the CD4 molecule rather than a viral protein, to deplete potential viral target cells irrespective of infection status. Despite profound CD4+ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not delayed in anti-CD4 treated animals compared with controls, likely due to the limited antibody-mediated cell depletion that occurred in rectal tissue and lymphoid follicles. Supported by ORIP (R24OD010976), NCI, and NIAID.