Whole genome analysis of CRISPR Cas9 sgRNA off-target homologies via an efficient computational algorithm

Background

The beauty and power of the genome editing mechanism, CRISPR Cas9 endonuclease system, lies in the fact that it is RNA-programmable such that Cas9 can be guided to any genomic loci complementary to a 20-nt RNA, single guide RNA (sgRNA), to cleave double stranded DNA, allowing the introduction of wanted mutations. Unfortunately, it has been reported repeatedly that the sgRNA can also guide Cas9 to off-target sites where the DNA sequence is homologous to sgRNA.

Results

Using human genome and Streptococcus pyogenes Cas9 (SpCas9) as an example, this article mathematically analyzed the probabilities of off-target homologies of sgRNAs and discovered that for large genome size such as human genome, potential off-target homologies are inevitable for sgRNA selection. A highly efficient computationl algorithm was developed for whole genome sgRNA design and off-target homology searches. By means of a dynamically constructed sequence-indexed database and a simplified sequence alignment method, this algorithm achieves very high efficiency while guaranteeing the identification of all existing potential off-target homologies. Via this algorithm, 1,876,775 sgRNAs were designed for the 19,153 human mRNA genes and only two sgRNAs were found to be free of off-target homology.

Conclusions

By means of the novel and efficient sgRNA homology search algorithm introduced in this article, genome wide sgRNA design and off-target analysis were conducted and the results confirmed the mathematical analysis that for a sgRNA sequence, it is almost impossible to escape potential off-target homologies. Future innovations on the CRISPR Cas9 gene editing technology need to focus on how to eliminate the Cas9 off-target activity.

     

基于结构的CRISPR蛋白xCas9的优化设计

酿脓链球菌 (Streptococcus pyogenes Cas9,SpCas9) 已成为强大的基因组编辑工具,但其可识别的前间隔序列临近基序 (Protospacer adjacent motifs,PAMs) 范围有限,且存在脱靶效应。为解决这些问题,文中提出一种对SpCas9的定向进化突变体xCas9进行优化的理性方法。首先,使用Rosetta程序进行能量最小化以优化Cas9的三维结构,获得其能量最低的构象;然后,对其定向进化所得氨基酸位点进行组合突变设计;最后,通过自由能排序从设计突变体中筛选出用于实验验证的最优变体。经DNA剪切实验验证,成功地获得了一个多PAM识别和低脱靶的新变体yCas9 (262A/324R/409N/480K/543D/694L/1219T)。该变体可识别NG、GAA和GAT序列,且其由错配sgRNA引导的脱靶DNA剪切活性低,为生物医学领域提供了一个有潜在应用价值的基因编辑工具。同时,文中还对SpCas9、xCas9和yCas9进行了分子动力学模拟,揭示了其PAM识别和脱靶效应的机理,可为进一步的CRISPR/Cas9蛋白优化改造提供理论指导。     

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.     

Increasing the efficiency of CRISPR‐Cas9‐VQR precise genome editing in rice

Clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR‐Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR‐Cas9‐VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.     

CRISPR/Cas9 activity in the rice <Emphasis Type="Italic">OsBEIIb</Emphasis> gene does not induce off-target effects in the closely related paralog <Emphasis Type="Italic">OsBEIIa</Emphasis>

Genome editing with the CRISPR/Cas9 system allows mutations to be induced at any 20-bp target site in the genome preceded by the short protospacer adjacent motif (PAM) 5′-NGG-3′. The brevity and degeneracy of the PAM ensures that the motif occurs every ~10 bp in plant genomes, and all plant genes therefore contain many targetable sites. However, the CRISPR/Cas9 system tolerates up to three mismatches in the target site, so the ability to target genes in a specific manner requires the design of synthetic guide RNAs (sgRNAs) that do not bind off-target sites anywhere else in the genome. This is straightforward for single-copy genes but more challenging if a target gene has one or more paralogs because the principles that balance targeting efficiency (the frequency of on-target mutations) and accuracy (the absence of off-target mutations) are not fully understood and may be partially species-dependent. To investigate this phenomenon in rice, we targeted the rice starch branching enzyme IIb gene (OsBEIIb) with two sgRNAs designed to differ at two and six positions, respectively, from corresponding sites in the close paralog OsBEIIa. In each case, half of the mismatches were in the essential seed region immediately upstream of the PAM, where exact pairing is thought to be necessary, and the other half were in the distal part of the target. The sgRNAs also differed in predicted targeting efficiency (39 and 96 %, respectively). We found that the sgRNA with the low predicted efficiency was actually the most efficient in practice, achieving a mutation frequency of 5 % at the target site, whereas the sgRNA with the high predicted efficiency generated no mutations at the second target site. Furthermore, neither of the sgRNAs induced an off-target mutation in the OsBEIIa gene. Our data indicate that efficiency predictions should be tested empirically because they do not always reflect the experimental outcome and that a 1-bp mismatch in the seed region of a sgRNA is sufficient to avoid off-target effects even in closely related rice genes.     

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR–Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR–Cas9 editing, raising an urgent issue to be addressed.     

<Emphasis Type="Italic">Arabidopsis glutamate:glyoxylate aminotransferase 1</Emphasis> (Ler) mutants generated by CRISPR/Cas9 and their characteristics

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) technology provides an efficient tool for editing the genomes of plants, animals and microorganisms. Glutamate:glyoxylate aminotransferase 1 (GGAT1) is a key enzyme in the photorespiration pathway; however, its regulation mechanism is largely unknown. Given that EMS-mutagenized ggat1 (Col-0 background) M2 pools have been generated, ggat1 (Ler background) should be very useful in the positional cloning of suppressor and/or enhancer genes of GGAT1. Unfortunately, such ggat1 (Ler) mutants are not currently available. In this study, CRISPR/Cas9 was used to generate ggat1 (Ler) mutants. Two GGAT1 target single-guide RNAs (sgRNAs) were constructed into pYLCRISPR/Cas9P_35S-N, and flowering Arabidopsis (Ler) plants were transformed using an Agrobacterium tumefaciens-mediated floral dip protocol. Eleven chimeric and two heterozygous GGAT1-edited T1 lines of target 1 were separately screened from positive transgenic lines. Two ggat1 homozygous mutants, CTC-deletion and T-deletion at target 1, were generated from T2 generations of the 13 T1 lines. The edited mutation sites were found to be stable through generations regardless of whether the T-DNA was present. In addition, the genetic segregation of the mutation sites obeyed the Mendelian single gene segregation rule, and no mutations were detected at the possible off-target site. Also, the two independent ggat1 mutants had similar photorespiration phenotypes and down-regulated GGAT enzyme activity. Together, these results indicate that genetically stable ggat1 (Ler) mutants were generated by CRISPR/Cas9 genome editing, and these mutants will be used to promote the positional cloning of suppressor and/or enhancer genes of GGAT1 in our subsequent study.     

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.     

CRISPR/Cas9系统中sgRNA设计与脱靶效应评估

基于CRISPR/Cas9系统介导的第三代基因组编辑技术,已成功应用于动物、植物和微生物等诸多物种的基因组改造。如何提高CRISPR/Cas9技术的基因组编辑效率和最大限度降低脱靶风险一直是本领域的研究热点,而使用高效且特异的sgRNA(Small guide RNA)是基因组改造成功的关键性因素之一。目前,已有多款针对CRISPR/Cas9技术的sgRNA设计和/或脱靶效应评估软件,但不同的软件各有优缺点。本文重点对16款sgRNA 设计和脱靶效应评估在线和单机版软件的特点进行了阐述,通过制定38项评估指标对不同软件进行了比较分析,最后对11种用于检测基因组编辑效率和脱靶的实验方法,以及如何筛选高效且特异的sgRNA进行了归纳总结。     

CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity

CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.     

Expanding the scope of CRISPR/Cas9‐mediated genome editing in plants using an xCas9 and Cas9‐NG hybrid

The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome‐editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9‐NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9‐NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG‐Cas9) based on mutations from both xCas9 and Cas9‐NG, and found that XNG‐Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG‐Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9‐NG) active in plant.     

机器学习方法在CRISPR/Cas9系统中的应用

基于CRISPR/Cas9系统介导的第三代基因组定点编辑技术,已被广泛应用于基因编辑和基因表达调控等研究领域。如何提高该技术对基因组编辑的效率与特异性、最大限度降低脱靶风险一直是该领域的难点。近年来,机器学习为解决CRISPR/Cas9系统所面临的问题提供了新思路,基于机器学习的CRISPR/Cas9系统已逐渐成为研究热点。本文阐述了CRISPR/Cas9的作用机理,总结了现阶段该技术面临的基因组编辑效率低、存在潜在的脱靶效应、前间区序列邻近基序(PAM)限制识别序列等问题,最后对机器学习应用于优化设计高效向导RNA (sgRNA)序列、预测sgRNA的活性、脱靶效应评估、基因敲除、高通量功能基因筛选等领域的研究现状与发展前景进行了展望,以期为基因组编辑领域的研究提供参考。     

新生儿短肠综合征的肠内营养研究进展

从喂养方式、喂养过程、营养成分、护理重点等多方面综述新生儿短肠综合征肠内营养的研究进展,并结合患儿的实际病情以及实验室检查结果等指标,提出对患儿进行持续性的肠内营养支持的具体做法：（1）在现实情况允许的条件下,保证用母乳喂养患儿,无法提供母乳的情况下合理配比奶粉,并根据实际需要添加一些纤维和脂类的补充剂,保障患儿健康发育;（2）在给予肠内营养的过程中全程无菌化处理,调节适宜的温度,合理选择药物;（3）应用大规模对照试验方式,通过大数据对比总结出持续肠内营养对短肠综合征患儿的影响,为儿科护理工作提供科学依据。     

Cas9HF1 enhanced specificity in Ustilago maydis

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.     

Recent advances in CRISPR/Cas9 mediated genome editing in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.     

<Emphasis Type="Italic">CAR1</Emphasis> deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98 % decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.     

CRISPR/Cas9基因编辑技术在丝状真菌中的应用

随着对丝状真菌基因水平研究的不断深入,CRISPR/Cas9技术作为先进的基因编辑技术,已被广泛应用于丝状真菌的基因编辑。探究了CRISPR/Cas9系统在不同丝状真菌中的应用情况,主要从sgRNA的构建与表达、Cas9蛋白的改造与表达、不同的DNA双链断裂修复（DNA double-strand break,DSB）方式等方面进行概述,并对编辑效率、脱靶效应进行总结,旨在为今后丝状真菌中CRISPR/Cas9系统的构建及改良提供思路。     

CRISPR/Cas9的应用及脱靶效应研究进展

CRISPR/Cas9基因编辑技术在生命科学领域掀起了一场全新的技术革命,该技术可以对基因组特定位点进行靶向编辑,包括缺失、插入、修复等。CRISPR/Cas9比锌指核酸酶 (ZFNs)和转录激活因子样效应物核酸酶(TALENs)技术更易于操作,而且更高效。CRISPR/Cas9系统中的向导RNA(Single guide RNA, sgRNA)是一段与目标DNA片段匹配的RNA序列,指导Cas9蛋白对基因组进行识别。研究发现,设计的sgRNA会与非靶点DNA序列错配,引入非预期的基因突变,即脱靶效应(Off-target effects)。脱靶效应严重制约了CRISPR/Cas9基因编辑技术的广泛应用。为了避免脱靶效应,研究者对影响脱靶效应的因素进行了系统研究并提出了许多降低脱靶效应的方法。文章总结了CRISPR/Cas9系统的应用及脱靶效应研究进展,以期为相关领域的工作提供参考。