Krishanu Saha, Ph.D.'s picture
Saha, Ph.D.
Assistant Professor
Departments of Biomedical Engineering and Medical History & Bioethics
Wisconsin Institute for Discovery

University of Wisconsin-Madison


Krishanu Saha is an Assistant Professor in the Department of Biomedical Engineering at the University of Wisconsin-Madison. He is also a member of the Wisconsin Institute for Discovery (WID). Prior to his arrival in Madison, Dr. Saha studied Chemical Engineering at Cornell University and at the University of California in Berkeley. In 2007 he became a Society in Science: Branco-Weiss fellow in the laboratory of Professor Rudolf Jaenisch at the Whitehead Institute for Biomedical Research at MIT and in the Science and Technology Studies program at Harvard University with Professor Sheila Jasanoff in Cambridge, Massachusetts. At UW-Madison, his lab is now funded by the NIH, NSF and EPA to perform research on pluripotent stem cells, regenerative medicine, disease modeling and synthetic biology. His lab has developed a wide array of engineering approaches that seek to generate new cells, organoids and tissues from patient samples, as well as a suite of gene-editing technologies to knockout, correct or insert transgenes into human cells. He is a Member of the Forum on Regenerative Medicine organized by the National Academies of Sciences, Engineering and Medicine and the Leadership Team of the NSF Center for Cell Manufacturing Technologies (CMaT).

Title of Abstract

Precision Nanomedicines Using Controlled Nonviral Assembly Around CRISPR-Cas9 Ribonucleoproteins

Presenting Author

Krishanu Saha


CRISPR ribonucleoproteins (RNPs) can generate programmable gene edits, however imprecise editing and efficient delivery to human cells are key challenges. Here we describe novel biochemical techniques to assemble various biomolecules and coatings with nanoscale precision around a RNP. First, by modifying the single guide RNA (sgRNA) with a short "S1m" RNA aptamer, we developed a modular strategy, termed a “S1mplex,” to assemble Cas9 RNPs with up to three biotinylated moieties. Using S1mplexes with biotinylated short oligonucleotides greatly improves the ratio of precise to imprecise editing by up to 18-fold over conventional methods, while assembly with fluorescent molecules allows selection and enrichment for cells with multiplexed gene edits. Second, we developed synthetic coatings for nonviral delivery of RNPs to mammalian cells. The “nanocapsule” coating strategy encapsulates a single Cas9 RNP into a novel cell-degradable thin polymeric shell that can be decorated with cell targeting ligands and other biomolecules. Nanocapsules frequently outperformed commercial cationic delivery reagents, while having significantly lower toxicity and higher stability. In human pluripotent stem cells in vitro and via subretinal injection into mice in vivo, robust gene editing (up to 25%) is observed with nanocapsules. The “polyplex” coating strategy is a pre-polymerized cationic polymer that coats and assembles with S1mplexes and nucleic acids. Polyplexes also have improved cytotoxic properties and enable nonviral delivery of CRISPR machinery with high levels of precise gene edits (up to 38%). Combined, these platform technologies - which utilize chemically-defined, off-the-shelf reagents - have significant promise for regenerative medicine applications in vitro (e.g., drug discovery, disease modeling) and in vivo (e.g., somatic gene editing).

Research Interests

The Saha lab is focused on human cell engineering. Our research generates new technologies and tools for genome engineering and epigenetic reprogramming, with an ultimate aim of building in synthetic functionality into human cells and bodies. Current projects involve cell reprogramming, somatic gene editing, ex vivo gene editing and engineered T cell therapy.

Author Listing

Amr A. Abdeen (1); Guojun Chen (1,2); Jared Carlson-Stevermer (1,3); Yuyuan Wang (1); Pawan Shahi (4); Ruosen Xie (1); Lucille Kohlenberg (1); Madelyn Goedland (1); Kaivalya Molugu (1); Meng Lou (1); Bikash Pattnaik (3,4); Shaoqin Gong (1,2,3,6); Krishanu Saha (1,3)


University of Wisconsin-Madison

All Author Affiliations

Wisconsin Institute for Discovery, University of Wisconsin-Madison (1); Materials Science and Engineering, University of Wisconsin–Madison (2); Biomedical Engineering, University of Wisconsin-Madison (3); Pediatrics, University of Wisconsin-Madison (4); Ophthalmology and Visual Sciences, University of Wisconsin-Madison (5); Chemistry, University of Wisconsin–Madison (6)