A Primer on CRISPR,and Adaptations for the Classroom

Jason M. PetersCarol Gross Lab

University of California, San Francisco

Outline

Introduction to CRISPR

Genome Editing and Ethics

CRISPR in the Lab: Identifying Drug Targets

CRISPR in the Classroom: Activities for Students

Fundamental Processes in Biology

http://www.yourgenome.org/

Discovery of CRISPR

Ishino et al., 1987, J. Bacteriol.; Jansen et al., OMICS, 2002

repeated sequence “spacers”

“The biological significance of these sequences is not known.”

CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats

Discovery of CRISPR

Barrangou et al., 2007, Science

CRISPR: Adaptive Immunity for Bacteria

Hsu et al., 2014, Cell

Cas9

gRNA

How Cas9 Finds It’s Target DNA

Sternberg et al., 2014, Nature

https://www.youtube.com/watch?v=M739wgbcKuA

CRISPR Editing

Jinek et al., 2012, Science; Cong et al., 2013, Science

DNA

Cas9

gRNADouble-strand

break

NHEJ

Gene inactivated

HRawesome thing!

Awesome thing in genome!

awesome thing!+Editing template

CRISPR Editing: Why is it so Powerful?

CRISPR is cheap, quick, and easy to use.

CRISPR Editing: Ethical Concerns

March 19, 2015; New York Times

April 1, 2015; Protein and Cell

April 3, 2015; Science

CRISPR Editing: Already Here

CRISPR Interference

CRISPRi: Reducing Gene Expression

Qi et al., 2013, Cell

CRISPRi

VIABLE

antibiotic (sub-MIC)

Functional Analysis of Essential Genes

knockout knockdown (CRISPRi)

product of anessential gene

DEAD DEAD

Comprehensive Analysis of Essential Genes

Peters et al., 2016, Cell

CRISPRi Antibiotic Target Discovery

Essential Gene Knockdown Library- antibiotic

Essential Gene Knockdown Library+ sub-MIC antibiotic (Trimethoprim)

direct target (DfrA)

on pathway

Peters et al., 2016, Cell

MAC-0170636 Targets UppS

Peters et al., 2016, Cell

CRISPR in the Classroom: SFSU

Student level: masters degree class in biotechnology

Goal: demonstrate targeted CRISPRi gene knockdown

Method: target red fluorescent protein in Bacillus subtilis, quantify knockdown using flow cytometry

Lily ChenNot student data!!!

CRISPR in the Classroom: High School?

Student level: entry level biology student (after lessons on DNA/RNA), or AP biology

Goal: demonstrate targeted CRISPRi gene knockdown

Method: target gene encoding sugar fermentation enzyme (e.g., lacZ) in Escherichia coli, observe knockdown using MacConkey/X-gal agar plates

Lac+ Lac- Lac+ (blue)

Summary

CRISPR is a powerful new tool for genome editing and control of gene expression

Ethical issues regarding the use of CRISPR technology (especially in humans) have yet to be resolved.

CRISPR is opening up new avenues of study in the field of biology

CRISPR can be adapted to the classroom, likely for students with varying experience levels