۱- A brief history of CRISPR

۲- CRISPR/Cas9: biology, functionality, and applications

۳- Therapeutic targets for ex vivo cell-based CRISPR/Cas9 genome editing

۴- CRISPR therapy in clinical trials

۵- Challenges and opportunities

References

Abstract

The recently developed CRISPR/Cas9 technology has revolutionized the genome engineering field. Since 2016, increasing number of studies regarding CRISPR therapeutics have entered clinical trials, most of which are focusing on the ex vivo genome editing. In this review, we highlight the ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications. In these studies, CRISPR/Cas9 tools were used to edit cells in vitro and the successfully edited cells were considered as therapeutics, which can be introduced into patients to treat diseases. Considering a large number of previous reviews have been focused on the CRISPR/Cas9 delivery methods and materials, this review provides a different perspective, by mainly introducing the targeted conditions and design strategies for ex vivo CRISPR/Cas9 therapeutics. Brief descriptions of the history, functionality, and applications of CRISPR/Cas9 systems will be introduced first, followed by the design strategies and most significant results from previous research that used ex vivo CRISPR/Cas9 genome editing for the treatment of conditions or diseases. The last part of this review includes general information about the status of CRISPR/Cas9 therapeutics in clinical trials. We also discuss some of the challenges as well as the opportunities in this research area.

A brief history of CRISPR

The term CRISPR, or the clustered regularly interspaced palindromic repeats, was first used by Jansen et al., in 2002 to describe a novel family of repetitive DNA sequences presented in the genomes of prokaryotes [1]. These unusual repeated sequences were first detected in 1987 by Nakata et al. in Escherichia coli [2], and then recognized to be widespread in archaea and bacteria by Mojica et al., in 2000 [3]. After Jansen et al. identified the CRISPR-associated (Cas) genes in 2002 [1], Mojica et al. [4], Vergnaud et al. [5], and Bolotin et al. [6] revealed the exrtachromosomal and phage-associated origins of the spacers that separate the individual direct repeats, in 2005. Speculations about CRISPR arrays as immune memory and defense mechanism against virus envisions were then proposed [6,7], while the first experimental evidence for CRISPR/Cas system based adaptive immunity was reported by Horvath et al., in 2007 [8]. Following the series discoveries on the basic function and mechanism of CRISPR systems [9–۱۲], in 2010 Moineau et al. demonstrated the Cas9 enzyme (formerly named Cas5, COG3513, Csn1 or Csx12), which is guided by spacer sequences, cleaves target DNA [13]. In 2012, Doudna, Charpentier et al. [14], and Siksnys et al. [15] proved that Streptococcus pyogenes and Streptococcus thermophiles Cas9 can be guided by CRSIPR RNAs (crRNAs) to cleave target DNA in vitro, through forming a double-strand break (DSB). In 2013, Zhang et al. [16], Church et al. [17], and Doudna et al. [18] independently demonstrated the applications of engineered CRISPR/Cas9 systems for genome editing in mammalian cells. This represented a giant leap forward in the genome engineering field and triggered a tremendous number of studies regarding the use of the CRISPR/Cas9 platform for eukaryotic gene editing in a wide range of species, including flies [19,20], zebrafish [21–۲۴], frogs [25], mice [26–۲۸], rats [26,29], pigs [30], and monkeys [31,32].