Humphrey and Kasinski Journal of Hematology & Oncology (2015) 8:31 DOI 10.1186/s13045-015-0127-3 JOURNAL OF HEMATOLOGY & ONCOLOGY REVIEW Open Access RNA-guided CRISPR-Cas technologies for genome-scale investigation of disease processes Sean E Humphrey and Andrea L Kasinski* Abstract From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells. This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes. In this review, we examine and compare recently developed CRISPR-Cas platforms for global genome editing and examine the advancements these platforms have made in guide RNA design, guide RNA/Cas9 interaction, on-target specificity, and target sequence selection. We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing. Introduction from a region of the host genome adjacent to the Clustered regularly interspaced short palindromic repeats CRISPR region is directed to the invading DNA in a (CRISPR)-Cas systems are adaptive immune systems used sequence-dependent manner via the crRNA. Once by many bacteria and archaea to fight off foreign DNA in bound to the foreign DNA, Cas9 introduces a double- the form of bacterial phages and/or plasmids [1-5]. Al- stranded break in the foreign DNA [11-13]. though multiple CRISPR-Cas types (I, II, and III) and sub- Realizing the potential power of a programmable nucle- types (I-A to I-F) have been identified in bacteria and ase to edit mammalian genomes, the CRISPR-Cas9 system archaea, we pay particular attention to the specifics of the has since been commercially developed as a technology type II since type II has been engineered and adapted for for use in loss-of-function (LOF) studies [13,14]. Regard- use in eukaryotic systems, which is the focus of this re- less of the platform, a tracrRNA, a mammalian optimized view. Generally, the CRISPR-Cas system works through Cas9 protein, and a small guide RNA (sgRNA) that is RNA-directed endonuclease cleavage of the invading analogous to the crRNA must be expressed at minimum. genomic sequence. The invading sequence is captured In some engineered systems, the sgRNA and the and inserted directly into the genome of the host organ- tracrRNA are expressed separately while in others, they ism between CRISPR regions (Figure 1A) [6-8]. Follow- are expressed as a single transcript [14-16]. The sgRNA ing foreign DNA infection, the sequences within the sequences are generally 20-bp long, but sgRNA sequences CRISPR regions are transcribed as a single RNA tran- of 17–18 bp are also effective [17]. Target sequences must script called a precursor CRISPR RNA (pre-crRNA). In be adjacent to a protospacer adjacent motif (PAM) se- the CRISPR-Cas9 system, the pre-crRNAs are bound by quence in the form of 5′-NGG (Figure 1B) [18-20]. Once additional RNAs termed transactivating CRISPR RNAs expressed in cells, the Cas9 protein, tracrRNA, and the (tracrRNAs) [9-12]. Once bound, the pre-crRNAs are sgRNA form a complex, bind to the target sequence, and processed into individual crRNA:tracrRNA duplexes by make a double-stranded break in the target. The break is RNase III and together form a complex with an endo- repaired via the cellular process of nonhomologous end nuclease [9-12]. The endonuclease Cas9 that is encoded joining (NHEJ), an error-prone process that introduces in- sertion, deletion, and frameshift mutations into the target sequence. Targeted mutations can also be introduced by * Correspondence: [email protected] cotransfecting single- or double-stranded oligodeoxynu- Department of Biological Sciences, Purdue University, 1203 West State Street, West Lafayette, IN 47907, USA cleotides to promote homology-driven repair [21-28]. © 2015 Humphrey and Kasinski; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Humphrey and Kasinski Journal of Hematology & Oncology (2015) 8:31 Page 2 of 9 Figure 1 The CRISPR-Cas9 bacterial immune system and design of a CRISPR-Cas9 target sequence. (A) The CRISPR-Cas system acts as an adaptive immune system in bacteria and archaea. Clustered regularly interspaced short palindromic repeats (CRISPR) regions are stretches of repetitive genomic bacterial or archaea DNA interspersed by segments of foreign DNA sequences captured from bacterial phages and plasmids. A cluster of Cas (CRISPR associated) genes are located near the CRISPR region. The Cas9 gene, which is unique to type II CRISPR systems, codes for an RNA-guided endonuclease. Following foreign DNA infection in type II CRISPR systems, the CRISPR region is transcribed as a single RNA transcript called a pre-crRNA, and in type II systems, the pre-crRNAs are bound by tracrRNAs, processed into individual crRNA:tracrRNA duplexes by RNase III and form a complex with Cas9. The crRNA sequences are complementary to the foreign DNA and direct the Cas9 nuclease to form a complex with the foreign DNA and introduce a double-stranded break. (B) CRISPR-Cas9 target sequences are 20-nt long and are flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG. In studying the molecular signaling pathways that im- for longitudinal studies, which can lead to confounding pinge on disease processes, many large- and small-scale off-target effects and false negatives [35]. Second, knock- expression studies of diseased tissues have provided ex- down by these methods can often be incomplete [36,37]. tensive lists of genes that are aberrantly expressed in dis- Gene knockout technologies, such as those mediated by eases such as cancer [29]. Such studies have greatly CRISPR-Cas9, overcome such deficits by knocking out in- advanced our knowledge about the gene expression sig- dividual gene expression at the genomic level and do so natures of disease and provided us with a wealth of with minimal off-target effects. And while RNAi is limited genes that are important predictive and prognostic bio- to inhibiting the function of RNAs, CRISPR-Cas9 technol- markers. The challenge moving forward is how to effect- ogy can be used to introduce random or targeted muta- ively separate the genes that are ‘drivers’ of disease from tions into any portion of the genome such as the coding gene ‘passengers’ whose aberrant expression has no rele- region, promoter, or enhancer regions of genes [38]. vance to the disease state. Loss-of-function studies are Importantly, the CRISPR-Cas9 system has recently an effective way to assess whether a gene is a driver of been developed into a tool for genome-scale loss-of- disease or a passenger. function screens by several laboratories. In this review, The CRISPR-Cas system has some important advantages we examine and compare these recently developed over other methods in LOF studies. At present, a widely genome-scale technologies. We also explore some of the used method for knocking down the expression of genes is potential therapeutic and innovative new uses of the through the induction of RNA interference (RNAi) either CRISPR-Cas technology beyond genome editing. through transfection or viral transduction of small interfer- ing RNAs (siRNA) or short hairpin RNAs (shRNA) Genome-scale CRISPR-Cas9 platforms (single Cas systems) [30-34]. While the use of RNAi has certainly advanced The various genome-scale CRISPR-Cas9 platforms target many fields, there are also some inherent drawbacks. First, genes by directing either a single Cas9 protein to each siRNAs and shRNAs must be continuously expressed targeted gene (referred to here as a single Cas system) or Humphrey and Kasinski Journal of Hematology & Oncology (2015) 8:31 Page 3 of 9 by directing two Cas9 proteins to each targeted gene (re- LentiGuide-Puro has a 100-fold increase in viral titer over ferred to here as a dual Cas system). Shalem and col- the original lentiCRISPRv1 vector. Also, the number of hu- leagues recently developed a single Cas lentiviral man genes targeted by the sgRNA library was increased to genome-scale CRISPR-Cas9 KnockOut (GeCKO) library 19,050, guide RNAs targeting 1,864 miRNAs were in- [39]. Each lentiviral vector in the library delivers stable cluded, and a sgRNA library against 20,611 mouse protein- expression of Cas9, a specific sgRNA generated against a coding genes and 1,175 miRNAs was also generated. single gene, and a puromycin selection marker to the Importantly, these powerful new reagents are available to cell. In total, 64,751 unique lentiviral vectors make up the academic community through Addgene. the GeCKO library that target 18,080 human genes. This A similar large-scale study also utilized the CRISPR- powerful new method of interrogating gene function on Cas9 system to develop a lentiviral library containing a genome-wide scale was used in both positive and nega- 73,000 sgRNAs against 7,114 genes that was used for tive selection screens in human cells. First, to test the genetic screens in human cells [41]. The library was ability of the GeCKO library to identify genes critical to transduced into cells stably expressing Cas9. This library survival, a negative selection screen was conducted by proved effective in positive and negative selection transducing a human melanoma cell line and a human screens at identifying genes important to the mismatch stem cell line with the GeCKO library.