Prashant Mali

University of California, San Diego

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Co-Authors

Kyle Ford

University of California, San Diego

J. Silvio Gutkind

University of California, San Diego

Nadia Arang

University of California, San Diego

Xingyu Wu

Qilu Hospital of Shandong University

Ayush Kishore

University of California, San Diego

Keiichi Koshizuka

University of California, San Diego

Michael M. Allevato

University of Michigan–Ann Arbor

Yusuke Goto

Chiba University

Hiroki Izumi

National Cancer Center Hospital East

Toshinori Ando

Hiroshima University Hospital

Cooperative Institutions

University of California, San Diego

195

Harvard University

Johns Hopkins University

Johns Hopkins Medicine

La Jolla Bioengineering Institute

Massachusetts Institute of Technology

University of Baltimore

University of California, San Francisco

La Jolla Alcohol Research

Sidney Kimmel Comprehensive Cancer Center

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Combinatorial CRISPR–Cas9 screens for de novo mapping of genetic interactions

Nature Methods (2017)

John Paul Shen Dongxin Zhao Roman Šášik Jens Luebeck Amanda Birmingham

Guide RNA

10.1038/nmeth.4225

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Citations (331)

Cas9 as a versatile tool for engineering biology

Nature Methods (2013)

Prashant Mali Kevin M. Esvelt George M. Church

Genome Engineering

Synthetic Biology

10.1038/nmeth.2649

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Citations (1,169)

Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas

iScience (2021)

Udit Parekh Daniella McDonald Amir Dailamy Yan Wu Thekla Cordes

Deconstructing tissue-specific effects of genes and variants on proliferation is critical to understanding cellular transformation and systematically selecting cancer therapeutics. This requires scalable methods for multiplexed genetic screens tracking fitness across time, across lineages, and in a suitable niche, since physiological cues influence functional differences. Towards this, we present an approach, coupling single-cell cancer driver screens in teratomas with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serial reinjection. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. Additionally, mutant MEK1S218D/S222D provides a proliferative advantage in mesenchymal lineages like fibroblasts. Our method provides a powerful platform for multi-lineage longitudinal study of oncogenesis.

Lineage (genetic)

10.1016/j.isci.2021.103149

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Citations (3)

Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures

Biomaterials (2021)

Michael Hu Xin Yi Lei Jon D. Larson Melissa McAlonis Kyle Ford

Ex vivo

Biofabrication

Genome Engineering

10.1016/j.biomaterials.2021.121276

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Citations (6)

Supplementary Figures 1-6, Table 1B, 2, 4-6 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Joyce E. Ohm Prashant Mali Leander Van Neste David M. Berman Liang Liang

Supplementary Figures 1-6, Table 1B, 2, 4-6 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Reprogramming

Epigenome

Table (database)

10.1158/0008-5472.22384490

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Citations (0)

A Novel Engineered U7 Small Nuclear RNA Scaffold Greatly Increasesin vitroandin vivoADAR-Mediated Programmable RNA Base Editing

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Susan M Byrne Stephen Burleigh Robert Fragoza Yue Jiang Yiannis A. Savva

Custom RNA base editing using the endogenous human Adenosine Deaminase Acting on RNA (ADAR) enzyme presents a promising approach for precision therapeutics, alleviating concerns of permanent DNA damage or immunogenicity from foreign bacterial proteins such as CRISPR/Cas. ADAR can be directed to act on therapeutic RNA targets by antisense guide RNAs (gRNAs) that create a substrate for ADAR′s adenosine-to-inosine (effectively A-to-G) deamination activity. Delivery of gRNAs via a DNA expression construct provided by Adeno-Associated Virus (AAV) might allow life-long duration of the therapy. However, a major challenge for RNA editing using gene-encoded gRNAs and endogenous levels of ADAR is achieving sufficient gRNA activity inside cells, especially in therapeutic situations where AAV delivery may provide as low as one viral genome per cell. Here we show that embedding antisense gRNAs into a U7 small nuclear RNA (snRNA) framework and adding hnRNP A1 binding domains greatly increases the efficiency of custom RNA editing. This increased editing efficiency allows for detectable RNA editing from a single genomic insertion of gRNA construct per cell, which enabled a pooled library screen of 750+ gRNA variations to further improve the SmOPT U7 hairpin system. The screen revealed critical residues responsible for RNA editing and generated new SmOPT and U7 hairpin variants that further boosted RNA editing. The final design, combined with an improved synthetic U7 promoter, resulted in up to 76% targeted editing with a single integrated copy of construct per cell, representing a 10- to 100-fold increase over existing circular gRNA approaches. Using systemic in vivo AAV delivery, we achieved an unprecedented 75% RNA editing in the total brain of a mouse model of Hurler syndrome. Our novel SmOPT U7 system also improved published antisense oligos for DMD exon skipping, currently in clinical trials, by up to 25-fold in differentiated myoblasts, and therefore represents a universal scaffold for ADAR-based RNA editing as well as other antisense RNA therapies.

ADAR

Base (topology)

10.1101/2024.09.29.615721

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Citations (0)

A homing CRISPR mouse resource for barcoding and lineage tracing

bioRxiv (Cold Spring Harbor Laboratory) (2018)

Reza Kalhor Kian Kalhor Kathleen Leeper Amanda R. Graveline Prashant Mali

Abstract Cellular barcoding using nuclease-induced genetic mutations is an effective approach that is emerging for recording biological information, including developmental lineages. We have previously introduced the homing CRISPR system as a promising methodology for generating such barcodes with scalable diversity and without crosstalk. Here, we present a mouse line (MARC1) with multiple genomically-integrated and heritable homing guide RNAs (hgRNAs). We determine the genomic locations of these hgRNAs, their activity profiles during gestation, and the diversity of their mutants. We apply the line for unique barcoding of mouse embryos and differential barcoding of embryonic tissues. We conclude that this mouse line can address the unique challenges associated with in vivo barcoding in mammalian model organisms and is thus an enabling platform for recording and lineage tracing applications in a mammalian model system.

DNA Barcoding

Tracing

Lineage (genetic)

10.1101/280289

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Citations (7)

In vivo RNA editing of point mutations via RNA-guided adenosine deaminases

Nature Methods (2019)

Dhruva Katrekar Genghao Chen Dario Meluzzi Ashwin Ganesh Atharv Worlikar

ADAR

Nucleic acid structure

Guide RNA