Chemically Modified Nucleic Acids for CRISPR-Cas
Chemically modified nucleosides are useful for the enhancement of genome editing efficiency when using the CRISPR-Cas systems. Recently scientists studying the CRISP Cas system type II showed that artificially modified nucleic acids can be successfully incorporated into CRISPR guide RNA (gRNA).
Recently, Hendel et al. in 2015 reported the use of chemically modified and protected nucleoside phosphoramidites for the synthesis of single guide RNAs (sgRNAs) to enhance genome editing efficiency in human primary T cells, CD34+ hematopoietic stem and progenitor cells. The researchers argued that co-delivery of chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system. According to Hendel et al., this approach is a simple and effective way for the development of new genome editing methods. This technique is thought to potentially accelerate the development of a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
Hendel et al. used three different modifications for the synthesis of sgRNAs. The modified nucleosides 2’-O-methyl (M), 2’-O-methyl-3’-phosphorothioate (MS), and 2’-O-methyl-3’-thiophosphonoacetate (MSP) were incorporated as protected nucleoside phosphoramidites. However, other modified nucleosides, such as bridged nucleic acids, can be used as well. The modified nucleic acids were incorporated at the 5′ and 3′ terminal positions of the synthetic sgRNAs.
Figure 1: Structures of chemical modifications that can be incorporated into RNAs, in this case into sgRNAs. Structures of RNA dimers and modified RNA chimeras are shown.
Many scientists now use the CRISPR-Cas system for targeted gene editing. Because of its ease of use CRISPR-Cas appears to have become the gene editing tool of choice. The increasing number of papers investigating the CRISPR-Cas systems and their use illustrate this nicely. Figure 1 shows the number of published papers as a result of a Pubmed search.
Figure 2: Numbers of published CRISPR-Cas papers in Pubmed.
The CRISPR (clustered regularly interspaced short palindromic repeat) loci together with a diverse cassette of CRISPR-associated (Cas) genes provide a sophisticated adaptive immune system to bacteria and archaea. The pre-crRNA is encoded in the CRISPR locus. This locus consists of repeat and spacer sequences. In some cases, the repeat sequences fold into stem-loop structures. The spacer sequences originate from previously cell attacking invader DNA. CRISPR locus transcription starts from a leader region yielding the pre-crRNA. The pre-crRNA is processed to generate crRNAs. Each crRNA is specific for one invader. (Yingjun Li, Saifu Pan, Yan Zhang, Min Ren, Mingxia Feng, Nan Peng, Lanming Chen, Yun Xiang Liang, and Qunxin She; Harnessing Type I and Type III CRISPR-Cas systems for genome editing Nucl. Acids Res. first published online October 13, 2015 doi:10.1093/nar/gkv1044).
Figure 2: The CRISPR locus. The CRISPR locus encodes the pre-crRNA that consists of repeat and spacer sequences. Some of the repeat sequences fold into stem-loop structures. Spacer sequences are derived from invader DNA from previous cell attacks. CRISPR locus transcription starts from the leader region (black arrow) generating pre-crRNA. The pre-crRNA is subsequently processed into crRNAs, and each crRNA is specific for one invader. (Maier L-K, Fischer S, Stoll B, et al. The immune system of halophilic archaea. Mobile Genetic Elements. 2012;2(5):228-232. doi:10.4161/mge.22530).
The CRISPR Cas base immune defense proceeds in three stages:
(1) Adaptation | Invading nucleic acid of the invading element enters the cell. This is immediately recognized as a foreign element. A piece of the invader DNA (the protospacer) is selected and integrated into the CRISPR locus as a new spacer. The protospacer as part of the invading DNA sequence is called a spacer after integration into the CRISPR locus. Selection of a new spacer depends on the presence of a specific neighboring sequence, the protospacer adjacent motif (PAM). This has been shown to be the case for CRISPR-Cas systems type I and type II. |
(2) Expression | The CRISPR locus is expressed. A pre-crRNA is generated and subsequently processed to short crRNAs. Each crRNA is specific for a single invader sequence. |
(3) Interference | Cas proteins together with crRNA recognize the invader during the defense reaction. The spacer sequence of the crRNA form base pairs with the invader sequence from which it was derived and hybridizes with it. This makes the defense sequence specific. |
CRISPR-Cas immune systems are classified into three main types and eleven or more subtypes. All CRISPR-Cas systems operate through three stages: acquisition, CRISPR RNA (crRNA) biogenesis, and target interference. CRISPR-Cas based genome editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage. The Cas endonuclease facilitates the cleavage. CRISPR-derived RNAs (crRNAs) together with Cas proteins capture and degrade invading genetic materials in prokaryotes.