Prolonged inflammation in the injury site during spinal cord injury makes re-generation of nerve cell
connections difficult. So, in order to fully comprehend the factor that causes inflammation, we use
zebrafish as our model organism, which relies on blood-derived macrophages to recover from spinal
cord injury. designed an in vivo experiment for screening the immune system in larval zebrafish using
CRISPR CAS9 Technology to study how larval zebrafish use blood-derived macrophage that keeps
neutrophils and il1 b – pro-inflammatory cytokine expression up to date during spinal cord injury
recovery We discovered that “four genes – thfb 1a, tgfb 3, tnfa, and sparc” of sCrRNAs are highly
active and successfully regenerate the spinal cord in larval zebrafish. Finally, a CRISPR-based
phenotypic screen in larval zebrafish revealed that the thfb 1 gene plays an important role in
controlling inflammation during the recovery process.
So even though severed spinal-cord nerve connections do not heal in humans and other mammals, a
spinal cord injury may result in permanent paralysis. In contrast, zebrafish can recover from spinal
cord injury through an inflammation-controlled process mediated by macrophages, a type of immune
system cell. The precise mechanism by which macrophages aid spinal cord regeneration in zebrafish
remains unknown. Identifying macrophage-related genes involved in zebrafish spinal cord
regeneration as quickly as possible. CRISPR/Cas9 technology is used in the strategy, which allows
researchers to target and disrupt specific genes, revealing their function. This gene-specific targeting
is made possible by molecules known as synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs).
The new method was used to study spinal cord regeneration in larval zebrafish. A prescreening step in
which they tested over 350 sCrRNAs that target genes already known to potentially play an important
role in inflammation-related spinal cord regeneration was critical to the method. By introducing these
sCrRNAs into zebrafish, researchers were able to identify ten genes that, when disrupted, hampered
recovery from spinal cord injury.
The list was narrowed down to four genes that appear to be critical for the repair of severed spinal
nerve connections, thereby validating the novel method. Tgfb1, in particular, appears to play an
important signalling role in controlling inflammation during the healing process.
The new method and findings could contribute to a better understanding of zebrafish spinal cord
regeneration. The method could also be used to screen for genes involved in other biological
processes, according to the researchers.
SCI and Pro-inflammatory Cytokine Involved in Spinal Cord Regeneration:
Inflammation after SCI is complicated and orchestrated by many cell types and inflammatory
cytokines such as tumour necrosis factor-alpha (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6)
(IL-6) the most important pro-inflammatory cytokines in general are IL-1, IL-6, and TNF-. These
cytokines communicate via type I cytokine receptors (CCR1), which differ structurally from other
cytokine receptor types. They are essential for coordinating cell-mediated immune responses and for
modulating the immune system.
CRISPR stands for clustered regularly interspaced short palindromic repeats, which are DNA
sequences found in the genomes of prokaryotic organisms like bacteria and archaea.Sequences
derived from bacteriophage DNA fragments that previously infected the prokaryote.Detection and
destruction of DNA from similar bacteriophages during subsequent infections.these sequences play an
important role in prokaryotic antiviral (anti-phage) defence and provide a form of acquired immunity.
Cas 9 is a CRISPR-associated protein.
It is an enzyme that recognises and cleaves specific strands of DNA that are complementary to the
CRISPR sequence using CRISPR sequences as a guide.Cas9 enzymes and CRISPR sequences form
the foundation of the CRISPR-Cas9 technology, which can be used to edit genes within organisms.
Using CRISPR CAS 9 to Model Spinal Cord Injury
Cas9 restriction enzymes, sCrRNAs, TracrRNAs, and TracrRNAs were extracted. The restriction
enzyme recognition sequences on sCrRNAs are designed to overlap the Cas9 cut site. In vivo testing
involves injecting a 1:l mixture of sCrRNAs, TracrRNA, Cas9 enzymes (CrRNAs targeting exon 1),
and fast green dye into the yolk of single-stage cell embryos. CrRNAs targeting sites tgfb 1a, tgfb 3
were used to generate stable mutants, while tnfa, sparc, and cst 7 were targeted with respective strands
of sCrRNAs to obtain a larger deletion. identified our desired traits and crossed them with wild-type
genes. All spinal cord lesion tests were carried out on an F3 heterozygous incross.
Analytical Parameters to Determine Which Pro-inflammatory Cytokine is Responsible for Spinal
Cord Injury Regeneration:
Restriction fragment length polymorphisms (RFLP), allele sequencing, Hexb activity assay, and
immunohistochemistry on whole-mount larvae were all used in this study. After completed the spinal
cord injury and compound incubation procedures. Finally, performed a behavioural assay and RTPCR.
Discussion:
focused on tgfb 1a and tgfb3 using previously available information on cytokines involved in
inflammation. that tgfb1a, as a regulator of post-injury inflammation, can help us understand how
inflammation is resolved quickly to promote recovery. As a result, we conclude that immune response
is required for spinal cord regeneration. This discovery may aid researchers in developing drug
compounds to treat rare neurodegenerative diseases, as well as in improving the recovery state of
spinal cord injury patients