Construction of Lentiviral Vectors for CRISPR/Cas9 Editing

The plasmids used in this study, pLenti-FNLS-P2A-Puro (Addgene#110841-CBE) and pLenti-ABERA-P2A-Puro (Addgene#112675-ABE), were a gift from Lukas Dow (Zafra et al., 2018 (link)), and pMD2.G and psPAX2 (second-generation lentiviral packaging construct, Addgene #12259, 11260) were a gift from Didier Trono. The pLenti-ABE8e-puro vector was constructed by amplifying ABE8e from the TadA-8e V106W plasmid (a plasmid gifted from David Liu, Addgene#138495) (Richter et al., 2020 (link)) using the primers mentioned in Supplementary file 2. The amplified PCR product was then cloned into pLenti-ABERA-P2A-Puro backbone after digestion with BamH1 and Nhe1 by HIFI assembly (NEB). The gRNA sequence from lentiCRISPR V2 vector (a construct gifted from Feng Zhang, Addgene#52961) was removed by digestion with EcoR1 and Kpn1 enzyme (NEB). The digested plasmid was then re-ligated with NEB Ligase after a exonuclease treatment (NEB Exonuclease 1) to generate a lentiCRISPR V2.1 vector (Sanjana et al., 2014 (link)). The plasmids were isolated using NucleoBond Xtra Midi EF (Macherey-Nagel) according to the manufacturer’s instruction.

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Ravi N.S., Wienert B., Wyman S.K., Bell H.W., George A., Mahalingam G., Vu J.T., Prasad K., Bandlamudi B.P., Devaraju N., Rajendiran V., Syedbasha N., Pai A.A., Nakamura Y., Kurita R., Narayanasamy M., Balasubramanian P., Thangavel S., Marepally S., Velayudhan S.R., Srivastava A., DeWitt M.A., Crossley M., Corn J.E, & Mohankumar K.M. (2022). Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. eLife, 11, e65421.

Publication 2022

pLenti-FNLS-P2A-Puro pLenti-ABERA-P2A-Puro Exonuclease 1 NucleoBond Xtra Midi EF

Backbone Digestion Enzyme Exonuclease Exonuclease 1 Ligase Nhe1 Plasmids Primers Vector

Corresponding Organization :

Other organizations : Institute for Stem Cell Biology and Regenerative Medicine, Christian Medical College, Sree Chitra Thirunal Institute for Medical Sciences and Technology, Christian Medical College & Hospital, Innovative Genomics Institute, Gladstone Institutes, UNSW Sydney, University of California, Berkeley, Manipal Academy of Higher Education, RIKEN BioResource Research Center, Japanese Red Cross Society, Japan, University of California, Los Angeles

Top 5 similar protocols

Variable analysis

independent variables

PLenti-FNLS-P2A-Puro (Addgene#110841-CBE)
PLenti-ABERA-P2A-Puro (Addgene#112675-ABE)
PMD2.G
PsPAX2
PLenti-ABE8e-puro vector
TadA-8e V106W plasmid (Addgene#138495)
LentiCRISPR V2 vector (Addgene#52961)
LentiCRISPR V2.1 vector

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

Not specified

negative controls

Not specified

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