# Generation of immunological memory by CRISPR-Cas systems

> **NIH NIH DP1** · ROCKEFELLER UNIVERSITY · 2020 · $1,186,500

## Abstract

Project Summary
CRISPR-Cas loci consist of short DNA repeats separated by equally short sequences (known as spacers) that
match the genomes of prokaryotic viruses (phages) and plasmids and confer sequence-based immunity
against these elements. Immunity is mediated by small, antisense CRISPR RNA molecules (crRNAs) that are
transcribed from spacers and guide CRISPR-associated (Cas) nucleases to the invading nucleic acid for
cleavage and destruction. During my post-doctoral studies I pioneered the study of CRISPR-Cas systems to
establish the foundations of this bacterial immunity pathway. Using genetics, I determined that CRISPR-Cas
systems target DNA molecules in a sequence-specific manner, a study that was key to understand the
mechanisms of CRISPR immunity at the molecular level. This finding predicted the existence of RNA-
programmable Cas nucleases and their current applications to genome editing.
Upon plasmid or phage infection, CRISPR-Cas system incorporate new spacer sequences that match the
genome of the invader. This process records a memory of the infection that is subsequently used to generate
the crRNA guides for the Cas nucleases. While the molecular mechanisms behind the recognition and
cleavage of target sequences by the Cas nucleases are well understood, how the host can acquire new
spacers; i.e. the immunization phase of the CRISPR-Cas immune response, is still a mystery. In this proposal I
plan to study how the prokaryotic host acquires new spacer sequences from its invaders, using a combination
of molecular genetics and next-generation sequencing approaches. Fundamental questions such as (i) how
autoimmunity, or the acquisition of spacers from the host chromosome, is prevented; (ii) how fast is the
immunization process compared to the viral infectious cycle; (iii) which other cellular pathways, if any, assist
CRISPR immunization; (iv) how new spacer sequences are sampled from the invader's genome; and (v) how
the immunization process affects the evolution of the host population; are not yet answered. The proposed
studies will substantially advance our understanding of the molecular mechanisms underlying CRISPR-Cas
immunization and the impact that these loci have on the ecology and evolution of prokaryotes organisms that
harbor them. In addition, our experiments will require or allow us to engineer CRISPR-Cas systems that
perform spacer acquisition with high frequency. Such systems will facilitate the development of technologies
with applications that require the recording of specific cellular events into a specific genomic locus to enable
researchers following long cellular histories. Thus the proposed studies could provide new ground to exploit
CRISPR immunization for revolutionary biotechnological and/or therapeutic purposes.

## Key facts

- **NIH application ID:** 9991629
- **Project number:** 5DP1GM128184-04
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Luciano A Marraffini
- **Activity code:** DP1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $1,186,500
- **Award type:** 5
- **Project period:** 2017-09-30 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9991629

## Citation

> US National Institutes of Health, RePORTER application 9991629, Generation of immunological memory by CRISPR-Cas systems (5DP1GM128184-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9991629. Licensed CC0.

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