Evidences

~60 bp increase per new spacer

E.coli strains BL21AI lack Cas genes but have a CRISPR locus. Cas1-Cas2 was expressed from a plasmid pADAPT. When infected with a virus, PCR and western blot showed an addition of ~60 bp including a spacer and a new repeat

He Jianku 2018

HIV free babies - announced genome-edited babies at a summit in Hong Kong, they used Cas9-mediated editing of the germline, so changes were heritable.

Problems with this included; poor oversight, ethical violations, unknown long-term risks.

Consequences included; global condemnation, strengthened regulations, ethical re-evaluation of CRISPR use.

Heath et al 2023

published how prime editing was successful in correcting a causative mutation in chronic granulomatous disease, restoring myeloid function.

Bolt and White labs

Work from the Bolt and White labs showed that the first incision for integration is closest to the leader of CRISPR and IHF explains why.

The severe bending of the CRISPR leader by IHF protein, positions the leader-repeat DNA junction for engagement by Cas1. This explains why new spacers are always integrated at the leader side, preserving chronological order in the CRISPR array.

Dillard et al 2018

Fluorescent protein assays demonstrated that cascade interacts with Cas1-Cas2 in a DNA-independent manner and translocates with it forming the PAC. Cas1-Cas2 remains associated with target bound cascade and can displace protein roadblocks e.g Cas9 suggesting a mechanism for long-range spacer acquisition.

Hickman & Dyda 2015

Discovered that casposases carry out similar biochemical reactions as the CRISPR-Cas-Cas2 complex, but with opposite substrate specificities; casposases integrate specific sequences into random target sites, whereas CRISPR Cas1-Cas2 integrates essentially random sequences into specific sites in the CRISPR locus.

Krupovic et al

Explained the requirement of Cas2 for integration of shorter DNA fragments (spacers), and also suggests Cas2 protein may have also evolved from the ancestral casposon, although so far Cas2 genes have not been identified in casposons.

Hayes et al 2016

Explained that the glutamine wedge (Q287 in CasA) flips apart the first PAM-proximal base pair allowing the seed to pair in cascade initiates the directional target DNA strand unwinding for base pairing with crRNA.

used CyroEM

Hochstrasser et al 2014

It is unknown whether Cas3 rides with cascade or finds cascade later. Found evidence to suggest Cas3 binds CasA once CasA has found PAM. And CasA positions Cas3 adjacent to the PAM to ensures cleavage and directional DNA degredation.

used EM and biochemical assays

Jinek et al

CyroEM analysis showed apo-Cas9 is auto-inhibited as a nuclease, and the binding of tracrRNA-crRNA opens it up to make DNA binding surfaces and a channel - tracrRNA is needed for functional Cas9.

This revealed that Cas9 requires a dual‑RNA guide to function, enabling the later invention of the single‑guide RNA (sgRNA).

Deltcheva et al 2011

Revealed tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III. in vitro cleavage assays with purified RNase III.

Parkes et al 2024

Parkes et al. used the IDT Alt‑RTM CRISPR workflow to design sgRNAs targeting DDX49 exons in human U2OS‑derived cells, selecting crRNA spacers adjacent to valid NGG PAMs.
This demonstrates how modern CRISPR editing relies on computational sgRNA design tools to choose effective guides based on PAM proximity and exon targeting.

Yang et al 2016

Demonstrated that HR can be done using dual AAV system delivery of Cas9 and repair template, to correct a metabolic liver disease mutation affecting OTC enzymes, in newborn mice, achieving up to 20% correction in hepatocyte. This highlights newborn liver as an optimal window for durable CRISPR-based gene therapy.

Mitalipov et al 2017

CRISPR gene editing in human embryos cause chromosomal mayhem including large deletions and rearrangements - Mitalipov showed 40% of changes were caused by the phenomenon gene conversion, where DNA repair processes copy a sequence from one chromosome pair to heal the other.

CRISPR–Cas9 editing in human embryos often causes large‑scale genomic damage, and many ‘corrections’ are actually gene‑conversion events rather than true HDR, making DSB‑based editing unsafe for therapeutic use.

Gaudeli et al 2017

Gaudelli et al. evolved the E. coli TadA tRNA adenine deaminase into a DNA‑active enzyme, creating the first adenine base editor.

• created TadA variants that deaminate A → I in DNA

• Inosine is read as G, giving an A•T → G•C conversion

This enabled precise A→G base conversions in the genome, solving the lack of natural DNA adenine deaminases and expanding the CRISPR editing toolkit.

Chen et al 2018

harnessing trans-collateral for DETECTR

show that RNA-guided DNA binding unleashes robust, indiscriminate single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules, catalyzed by the same active site responsible for site-specific dsDNA cutting.

demonstrate that DETECTR enables rapid and specific detection of HPV in human patient samples, thereby providing a simple platform for nucleic acid-based, point-of-care diagnostics

Jennifer Doudna in 2018, Chen et al

discovered DETECTR, developed by chen et al

Zhang et al in 2017

Zhang et al. developed SHERLOCK, an isothermal diagnostic platform that uses Cas13a’s target‑activated collateral ssRNA cleavage to cut reporter molecules and generate fluorescent or lateral‑flow signals.
This established Cas13a collateral cleavage as a programmable molecular detection system, enabling rapid, sensitive diagnostics later adapted for COVID testing.

Joung et al 2020

developed STOP (SHERLOCK Testing in One Pot) for detecting SARS-CoV-2 in one hour that is suitable for point-of-care use.

STOPCovid represents a promising platform for developing POC COVID-19 diagnostics and has the potential to play an important role in effective test-trace-isolate measures to end the COVID-19 pandemic.

Zhang et al., 2017

CRISPR editing of butterfly wing‑pattern genes. Researchers knock out the optix gene using CRISPR‑Cas9, which disrupts red and orange pigmentation. The mechanism is simple: Cas9 creates a double‑strand break in the optix gene, the cell repairs it imperfectly, and the gene stops functioning. This allows scientists to test how colour patterns affect predator avoidance, mate choice, and camouflage in natural environments — directly linking a single gene to an ecological trait.

Wei et al., 2023

used CRISPR‑Cas12a environmental DNA (eDNA) assay that detects rare fish species directly from water samples with extremely high sensitivity. The system identifies target DNA using Cas12a’s collateral cleavage activity after recombinase polymerase amplification (RPA), enabling detection of as little as 6 copies of eDNA per µL within 35 minutes. This makes it a powerful tool for field‑based biodiversity monitoring and ecological surveillance.