What Is CRISPR?

CRISPR, short for clustered regularly interspaced short palindromic repeats, is a family of DNA sequences found in bacteria and archaea that forms the basis of an adaptive immune system, and, by extension, the genome editing technology derived from it. The natural system stores fragments of viral DNA between short repeated sequences, transcribes them into guide RNAs, and uses those guides to direct a CRISPR-associated nuclease to cut matching sequences in an invading genome. Repurposed as a laboratory tool, the same machinery lets researchers direct a cut to almost any chosen site in a genome by supplying a synthetic guide RNA, which placed CRISPR at the center of genetic engineering within a few years of its introduction.

The repeats were first noticed in Escherichia coli in 1987 and their immune function was demonstrated in the mid-2000s. The step that made the system a general-purpose tool came in 2012, when Jennifer Doudna, Emmanuelle Charpentier, and colleagues showed that the two RNAs guiding Cas9 could be fused into a single programmable guide. That work, summarized in their 2014 review of the new frontier of genome engineering with CRISPR-Cas9, earned Doudna and Charpentier the 2020 Nobel Prize in Chemistry.

How Targeted Editing Works

Cas9 binds a guide RNA roughly twenty nucleotides long and scans double-stranded DNA for a short protospacer adjacent motif, or PAM, which for the commonly used Streptococcus pyogenes enzyme is the sequence NGG. Where the guide matches the adjacent sequence, the nuclease creates a double-strand break. The cell then repairs that break by one of two competing pathways. Non-homologous end joining is error-prone and typically inserts or deletes a few bases, which is enough to knock out a gene. Homology-directed repair, which requires a supplied donor template and is active only in dividing cells, allows precise substitution of a sequence. Editing efficiency, the balance between these two repair pathways, and the frequency of off-target cuts at similar sequences elsewhere in the genome are the practical constraints on any experiment. The National Science Foundation account of how CRISPR reached the laboratory traces the route from basic research on bacterial immunity to that capability.

Base and Prime Editing

Double-strand breaks are blunt instruments, and the field has moved toward chemistries that avoid them. Base editors fuse a catalytically impaired Cas9 to a deaminase enzyme that chemically converts one base to another, changing C to T or A to G at a targeted position without cutting both strands. Prime editors couple a nickase to a reverse transcriptase and a guide RNA that carries the intended edit, writing new sequence directly into the target site and covering insertions, deletions, and all twelve base substitutions. Catalytically dead Cas9 fused to transcriptional activators or repressors gives CRISPR interference and activation, which modulate gene expression without altering sequence at all. Alternative nucleases including Cas12a and the RNA-targeting Cas13 broaden the PAM requirements and the substrate range.

Therapeutic and Diagnostic Translation

Clinical translation arrived quickly. Ex vivo approaches remove a patient's cells, edit them, and return them, an approach used in the first approved CRISPR therapy for sickle cell disease and transfusion-dependent beta thalassemia, which disrupts a repressor to reactivate fetal hemoglobin. In vivo delivery through lipid nanoparticles or adeno-associated virus vectors targets tissue directly, and a review of CRISPR technologies for in vivo and ex vivo gene editing surveys the delivery and safety questions that dominate this work. Cas13 and Cas12 systems also underpin rapid nucleic acid diagnostics, since their collateral cleavage activity can be read out on a lateral flow strip. Germline editing remains prohibited or restricted in most jurisdictions following the 2018 case of edited human embryos in China.

Applications

CRISPR has applications in a wide range of fields, including:

• Gene therapy for inherited blood, eye, and metabolic disorders
• Cancer immunotherapy and engineered T-cell production
• Crop improvement for yield, drought tolerance, and disease resistance
• Livestock breeding and disease resistance
• Rapid point-of-care molecular diagnostics
• Functional genomics screens and disease model construction