An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo.
The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia; Department of Clinical Haematology and Bone Marrow Transplant Service, the Royal Melbourne Hospital, Parkville, VIC 3050, Australia.
Brandon J
BJ
Aubrey
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
Gemma L
GL
Kelly
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
Andrew J
AJ
Kueh
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
Margs S
MS
Brennan
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
Liam
L
O'Connor
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
Liz
L
Milla
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
Stephen
S
Wilcox
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
Lin
L
Tai
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia.
Andreas
A
Strasser
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia. Electronic address: herold@wehi.edu.au.
Marco J
MJ
Herold
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D000072224:Bcl-2-Like Protein 11; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D059766:DNA End-Joining Repair; D004318:Doxycycline / Q000494:pharmacology; D020043:Genes, Essential / Q000235:genetics*; D052138:Genes, Neoplasm / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009154:Mutation; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*; D014183:Transplantation, Heterologous; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D000094704:RNA, Guide, CRISPR-Cas Systems; D016159:Tumor Suppressor Protein p53; D004318:Doxycycline