Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases

There are an estimated 10000 monogenic diseases affecting tens of millions of individuals worldwide.The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients.CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA.The complexity of genomic insults resulting in heritable disease requires patientspecific genome editing strategies with consideration of DNA repair pathways,and CRISPR/Cas systems of different types,species,and those with additional enzymatic capacity and/or delivery methods.In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair,non-homologous end joining,microhomology-mediated end joining,and base editing to permanently correct diverse monogenic diseases.

Combination of CRISPR/Cas9 System and CAR-T Cell Therapy:A New Era for Refractory and Relapsed Hematological Malignancies

Chimeric antigen receptor T(CAR-T)cell therapy is the novel treatment strategy for hematological malignancies such as acute lymphoblastic leukemia(ALL),lymphoma and multiple myeloma.However,treatment-related toxicities such as cytokine release syndrome(CRS)and immune effector cell-associated neurotoxicity syndrome(ICANS)have become significant hurdles to CAR-T treatment.Multiple strategies were established to alter the CAR structure on the genomic level to improve efficacy and reduce toxicities.Recently,the innovative gene-editing technology-clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease9(Cas9)system,which particularly exhibits preponderance in knock-in and knockout at specific sites,is widely utilized to manufacture CAR-T products.The application of CRISPR/Cas9 to CAR-T cell therapy has shown promising clinical results with minimal toxicity.In this review,we summarized the past achievements of CRISPR/Cas9 in CAR-T therapy and focused on the potential CAR-T targets.

CRISPR/Cas systems for the detection of nucleic acid and nonnucleic acid targets

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)systems are becoming powerful tools for disease biomarkers detection.Due to the specific recognition,cis-cleavage and nonspecific trans-cleavage capabilities,CRISPR/Cas systems have implemented the detection of nucleic acid targets(DNA and RNA)as well as non-nucleic acid targets(e.g.,proteins,exosomes,cells,and small molecules).In this review,we first summarize the principles and characteristics of various CRISPR/Cas systems,including CRISPR/Cas9,Cas12,Cas13 and Cas14 systems.Then,various types of applications of CRISPR/Cas systems used in detecting nucleic and non-nucleic acid targets are introduced emphatically.Finally,the prospects and challenges of their applications in biosensing are discussed.

Precise genome editing with base editors

Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potential and are now being actively pursued.The emerge of precise and effcient genome editing strategies such as base editing and prime editing provide powerful tools for nucleotide conversion without inducing double-stranded DNA breaks(DSBs),which have shown great potential for curing genetic disorders.A diverse toolkit of base editors has been devel-oped to improve the editing effciency and accuracy in different context of application.Here,we summarized the evolving of base editors(BEs),their limitations and future perspective of base editing-based therapeutic strategies.

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has rapidly been developed into a robust,multifunctional genome editing tool with many uses.Following the discovery of the initial CRISPR/Cas-based system,the technology has been advanced to facilitate a multitude of different functions.These include development as a base editor,prime editor,epigenetic editor,and CRISPR interference(CRISPRi)and CRISPR activator(CRISPRa)gene regulators.It can also be used for chromatin and RNA targeting and imaging.Its applications have proved revolutionary across numerous biological fields,especially in biomedical and agricultural improvement.As a diagnostic tool,CRISPR has been developed to aid the detection and screening of both human and plant diseases,and has even been applied during the current coronavirus disease 2019(COVID-19)pandemic.CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases,including cancers,and has aided drug development.In terms of agricultural breeding,precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins,starch,oil,and other functional components for crop improvement.Adding to this,CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators.Looking to the future,increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology.This review provides an in-depth overview of current CRISPR development,including the advantages and disadvantages of the technology,recent applications,and future considerations.

Mageb4,a Testis-Specific Gene,is Dispensable for Mouse Spermatogenesis

Objective:It has recently been shown that the melanoma antigen gene(MAGE)family is expressed in various tumor cell lines but silent in normal tissues,except germ cell lines.Mageb4,a member of the MAGE family,is highly expressed in the testis and homologous in humans and mice.Whole-exome sequencing studies have identifiedMageb4 as a possible X-linked cause of inherited male infertility.However,the function of Mageb4 protein remains largely unknown.Methods:Using clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)9 technology,we generated aMageb4 knockout mouse model(Mageb4^-/Y)to explore the role of this gene in spermatogenesis.Results:First,immunostaining of testicular cells showed thatMageb4 is localized in the cytoplasm of spermatogonia.Second,Mageb4^-/Y male mice displayed significant increases in apoptosis.However,Mageb4^-/Y male mice showed normal fertility,including normal sperm concentration,sperm motility,and testicular and epididymal histology.Conclusions:These findings suggest that,despite testis-exclusive expression,Mageb4 is dispensable for mouse spermatogenesis.Future research should focus on the role of this gene in apoptosis,aiming to provide clinical guidance regarding male infertility.