Jaspal S. Khillan, Ph.D.

Chief, Mouse Genetics and Gene Modification Section

Major Areas of Research

  • CRISPR/cas9 Genome editing in mouse embryos, ES cells and somatic cells
  • Generation of transgenic animal models
  • Development of embryonic stem (ES) and induced pluripotent stem (iPS) cell lines from different species 
  • Cryopreservation of germplasm for long term storage

Program Description

Genome editing via CRISPR cas9 has revolutionized the field of in vivo genome modification which has been used to achieve high editing efficiencies in a wide range of different cell types and embryos to create animal models. A small 20 base guide RNA can locate Cas9 endonuclease to the targeted site which causes the double stranded break (DSB) in the genomic DNA. The DSB is then filled with either non-specific non-homologous end joining (NHEJ), indels, or by more precise homology derived repair (HDR) in the presence of a template with homology sequences. MGGM provides CRISPR cas9 gene edited mouse models for infectious diseases, gene regulation and for specific human mutations. The Core possesses specialized expertise and state-of-the-art equipment for providing services for in vivo gene modification in mouse embryos for the production of transgenic, CRISPR mediated gene knockout (KO), gene knock-in (KI) and conditional gene KO mouse models to NIAID investigators. Gene KO and gene KI mice can also be generated via gene targeting in ES cells and chimera production. MGGM Core offers genome editing design (guide selection, donor DNA design, and genotyping), in addition to an on- and off-target mutagenesis genotyping service.

The MGGM Core also offers service for cryopreservation of mouse embryos and sperm for long-term storage, rederivation of mouse lines, in vitro fertilization (IVF) and mouse colony expansion.

Biography

Dr. Khillan obtained his Ph.D. from Punjab University Chandigurh India. He joined NIAID’s Comparative Medicine Branch (CMB) in 2015 and established the Mouse Genetics and Gene Modification (MGGM) section to provide state of the art technologies of mouse genome modification, including CRISPR/cas9 mediated genome editing, generation of transgenic mouse models, gene targeting in ES cells, iPS cells, germplasm cryopreservation and rederivation of mouse lines. He was the first to establish transgenic technology at NIH. His areas of research include development of ES and iPS cell lines from different species, gene regulation, creation of animal models for human genetic disorders, and CRISPR/cas9 genome editing in stem cells and somatic cells.

Selected Publications

Ernst O, Sun J, Lin B, Banoth B, Dorrington MG, Liang J, Schwarz B, Stromberg KA, Katz S, Vayttaden SJ, Bradfield CJ, Slepushkina N, Rice CM, Buehler E, Khillan JS, McVicar DW, Bosio CM, Bryant CE, Sutterwala FS, Martin SE, Lal-Nag M, Fraser IDC. A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation. Sci Signal. 2021 Aug;14(694):eabe0387. 

Longenecker G, Cho K, Khillan JS, Kulkarni AB. Cryopreservation Protocols for Genetically Engineered Mice. Curr Protoc. 2021 May;1(5):e138. 

Kline JM, Heusinkveld LE, Taranto E, Martin CB, Tomasi AG, Hsu IJ, Cho K, Khillan JS, Murphy PM, Pontejo SM. Structural and functional analysis of Ccr1l1, a Rodentia-restricted eosinophil-selective chemokine receptor homologue. J Biol Chem. 2021 Jan-Jun;296:100373. 

Mendenhall MA, Liu S, Portley MK, O'Mard D, Fattah R, Szabo R, Bugge TH, Khillan JS, Leppla SH, Moayeri M. Anthrax lethal factor cleaves regulatory subunits of phosphoinositide-3 kinase to contribute to toxin lethality. Nat Microbiol. 2020 Dec;5(12):1464-1471. 

Hall B, Cho A, Limaye A, Cho K, Khillan J, Kulkarni AB. Genome Editing in Mice Using CRISPR/Cas9 Technology. Curr Protoc Cell Biol. 2018 Dec;81(1):e57.

Visit PubMed for a complete publication listing.

Tools & Equipment

  • Overseeing tools of CRISPR genome editing 
  • Service to create CRISPR genome edited animals and cell lines
  • ES cell and iPS cells and CRISPR genome editing 
  • Service to generate transgenic animals and cryopreservation of sperm and embryos
  • Rederivation of SPF mouse lines
  • Database on cryopreservation of biomedical research animal models 

Contacts

Location

12735 Twinbrook Parkway
Twinbrook 3, Room 1E12C3
Rockville, MD 20852

Services

CRISPR cas9 genome edited mouse models

  • Generation of gene knockout mice via microinjection and electroporation
  • Generation of knock-in mice using single stranded oligodenucleotides (ssODNTs) 
  • Generation of gene knock-in mice via double stranded plasmid DNA or long single stranded DNA (lssDNA)




CRISPR/cas9 genome editing diagram

CRISPR/cas9 genome editing. Cas9 protein guided by sgRNA binds the DNA to cause double stranded break (DSB). The break is then filled by random bases via non-homologous end joining (NHEJ) or by homology derived repair in the presence of a template, oligo dinucleotide (ODN) with homology arms.

Credit
NIAID

CRISPR/cas9 genome editing. Cas9 protein guided by sgRNA binds the DNA to cause double stranded break (DSB). The break is then filled by random bases via non-homologous end joining (NHEJ) or by homology derived repair in the presence of a template, oligo dinucleotide (ODN) with homology arms.

Credit:
NIAID

Transgenic mouse models

  • Generation of transgenic mice via traditional DNA injection
  • Generation of transgenic mice via Bac injection




transgenic animals diagram

Generation of transgenic animals by DNA microinjection. The genomic DNA containing gene for green florescence protein (GFP), (top), is microinjected into the pronucleus of one cell embryo (middle) to generate pups that exhibit green florescence (bottom).

Credit
NIAID

Generation of transgenic animals by DNA microinjection. The genomic DNA containing gene for green florescence protein (GFP), (top), is microinjected into the pronucleus of one cell embryo (middle) to generate pups that exhibit green florescence (bottom).

Credit:
NIAID

Gene targeting in ES cells, iPS cells and somatic cells

  • Gene targeting in mouse ES cells and chimera production
  • CRISPR cas9 genome editing ES cells, iPS cells and somatic cells.




Microinjection of ES cells into Blastocyst.

Microinjection of ES cells into Blastocyst. Gene targeted ES cells (left panel) are microinjected into the cavity of expanded blastocyst using very fine needles to create chimeric animals for germline transmission of mutated gene.

Credit
NIAID

Microinjection of ES cells into Blastocyst. Gene targeted ES cells (left panel) are microinjected into the cavity of expanded blastocyst using very fine needles to create chimeric animals for germline transmission of mutated gene.

Credit:
NIAID

Cryopreservation of germplasm and rederivation of lines

  • Mouse sperm cryopreservation
  • Mouse embryo cryopreservation
  • In vitro fertilization (IVF) using mouse sperm
  • Rederivation of mouse lines from embryo and sperm




Sperm (left) and embryo cryopreservation (right).

Sperm (left) and embryo cryopreservation (right).

Credit
NIAID

Sperm (left) and embryo cryopreservation (right).

Credit:
NIAID

Training

  • Training in genome modification via CRISPR in embryos and stem cells
  • Training in generation of transgenic animals 
  • Training mouse embryo manipulation technologies

Research Group

 Caption: Mouse Genetics and Gene Modification Section, May 23, 2022: (left to right), Jessica Edwards, cryopreservation Expert, Cheng-Chao Lin, Molecular/cell Biologist, Jaspal S. Khillan, Chief, Megan Mallett, Cryopreservation Expert, Kyoungin Cho Molecular Biologist/Lab Manager, Katina Krasneck, Molecular Biologist.

Caption: Mouse Genetics and Gene Modification Section, May 23, 2022: (left to right), Jessica Edwards, cryopreservation Expert, Cheng-Chao Lin, Molecular/cell Biologist, Jaspal S. Khillan, Chief, Megan Mallett, Cryopreservation Expert, Kyoungin Cho Molecular Biologist/Lab Manager, Katina Krasneck, Molecular Biologist.

Credit
NIAID

Caption: Mouse Genetics and Gene Modification Section, May 23, 2022: (left to right), Jessica Edwards, cryopreservation Expert, Cheng-Chao Lin, Molecular/cell Biologist, Jaspal S. Khillan, Chief, Megan Mallett, Cryopreservation Expert, Kyoungin Cho Molecular Biologist/Lab Manager, Katina Krasneck, Molecular Biologist.

Credit: NIAID

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