CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury

Abstract

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

Targeting GPCRs & their Signaling as a Therapeutic Option in Melanoma

“…Zebrafish may be a suitable model for these types of studies. Indeed, it is relatively easy to generate and maintain large groups of transgenic animals [298][299][300][301]. Furthermore, zebrafish express many of the GPCRs expressed in humans, as well as their signaling machinery [302,303].…”

Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish

“…While dgRNP-based mutagenesis has been shown to efficiently disrupt gene function in F0 zebrafish even as single dgRNP injections (Kotani et al, 2015;Hoshijima et al, 2019;Davis et al, 2021;Keatinge et al, 2021;Klatt Shaw and Mokalled, 2021;Kroll et al, 2021;Zhao et al, 2021), it is becoming clear that anticipated phenotypic detections can depend on target genes and the mutagenic ability of each designed crRNA. This is likely due to varied genetic compensatory responses to mutations in individual loci (Kok et al, 2015;Rossi et al, 2015;El-Brolosy et al, 2019;Hoshijima et al, 2019) and the variable efficiency of synthetic crRNAs in gene editing (Keatinge et al, 2021;Kroll et al, 2021). Consistent with these prior reports, we noted that single dgRNP injections often led to variable phenotypes, with only some exhibiting high potency in our experiments (Figure 5G).…”

“…Many studies have reported efficient methods for generating F0 zebrafish mutants using in vitro-transcribed guide RNA (gRNA; Jao et al, 2013;Shah et al, 2015;Varshney et al, 2015;Burger et al, 2016;Tsarouchas et al, 2018;Wu et al, 2018;Xu et al, 2020), or more recently, using a dual-guide CRISPR/Cas9 ribonucleoprotein (dgRNP) system that utilizes a chemically synthesized duplex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA; Kotani et al, 2015;Hoshijima et al, 2019;Davis et al, 2021;Keatinge et al, 2021;Klatt Shaw and Mokalled, 2021;Kroll et al, 2021;Parab et al, 2021;Zhao et al, 2021). However, there is currently no integrated, standardized approach that is well established in the community, making it difficult to select which method to utilize based on pre-existing and emerging information in the rapidly evolving field.…”

“…This finding reported by Hoshijima et al in late 2019 has allowed the broader use of a synthetic crRNA-based dgRNP method as a potentially more efficient genome editing approach. However, despite the higher efficacy of synthetic crRNAs at cleaving target sequences in general, their ability to induce monoallelic or biallelic gene inactivation in F0 animals depends on the target sequences of individual crRNAs, since variable efficiency of crRNAs in gene editing has been reported (Keatinge et al, 2021). To circumvent this variable effect of individual crRNAs, a recent study injected an increasing number of dgRNPs per gene and showed enhanced efficiency of biallelic inactivation (Kroll et al, 2021).…”

Insights Into Central Nervous System Glial Cell Formation and Function From Zebrafish

“…The efficiency of new reverse genetic technology is continually improving, enabling more rapid investigation of genes for glial function. One example of this is the use of CRISPR/Cas9 strategies that can now be utilised to reliably induce somatic mutations with high efficiency in F0 embryos, called “crispants.” These significantly increase the speed with which genetic manipulations can be investigated ( Keatinge et al, 2021 ; Klatt Shaw and Mokalled, 2021 ; Kroll et al, 2021 ), which can allow the investigation of duplicated genes, those with potentially redundant functions and even multiple pathways in parallel. From a practical view, these technologies reduce the personnel and space requirements of mutagenesis-based forward genetic screens, thereby allowing scalable investigation of gene function by most laboratories.…”